Diagnostic kits for detection of target nucleic acid sequences

ABSTRACT

The present invention makes possible the catalytic production of sequence specific oligonucleotides through the use of a specially designed template sequence. The reaction can be made to proceed isothermally in the presence of an excess of nucleoside triphosphates, an agent for polymerization, and a cutting agent. Because the process is catalytic with respect to the template sequence, an unlimited amount of oligonucleotide product can theoretically be generated from a single molecule of template. Where the process is initiated by the presence of a nucleic acid target sequence, the method of the present invention can be used for diagnostic purposes as an amplification method to improve sensitivity. Diagnostic sensitivity can be further enhanced by employing a cascade of these template sequences.

This application is a continuation of application Ser. No. 08/200,897,filed Feb. 23, 1994, now U.S. Pat. No. 5,645,987, which is acontinuation of application Ser. No. 08/050,712 filed Apr. 19, 1993,abandoned, which is a continuation of application Ser. No. 07/762,140filed Sep. 23, 1991, abandoned, which is a continuation-in-part ofapplication Ser. No. 07/586,368 filed Sep. 21, 1990, abandoned, whichapplications are all hereby incorporated by reference.

BACKGROUND

Sequence specific oligonucleotides are used extensively in the fields ofmolecular biology and probe diagnostics for such varied purposes as, forexample, site directed mutagenesis, gene assembly, and as DNA probes toidentify the presence of specific nucleic acid sequences in patient testsamples. In addition, oligonucleotide probe mixtures have been preparedin research laboratories, based on the protein sequence of a resultinggene product and the degeneracy of the DNA code, to identify andcharacterize the nucleic acid sequences responsible for encoding thesegene products. Even more recently, research has been directed toward theuse of oligonucleotide sequences in a field which has become known as"antisense" therapy, whereby the oligonucleotides act as therapeuticagents to combat human diseases caused by viral and bacterial pathogens.

Oligonucleotide products have been generated in a number of differentways including both organic synthesis techniques and traditional cloningprocedures. Organic synthesis techniques have been optimized to achievemoderate quantities of oligonucleotides. These synthetic methods includetechniques referred to as the di-ester, the tri-ester, theH-phosphonate, and the phosphoramidite methodologies, with the solidsupport synthesis of oligonucleotides by the phosphoramidite methodbeing the most widely employed. A review of current methods ofoligonucleotide synthesis is provided in Caruthers, ch. 1,Oligonucleotides, Antisense Inhibitors of Gene Expression, ed., Cohen,CRC Press, Inc., Boca Rotan, Fla. (1989). Currently availabletechnologies and instrumentation are, however, still limited withrespect to the quantities of the synthetic oligonucleotide products thatcan be generated. In addition, the relatively harsh chemistries of thesesynthetic methods can produce a significant percentage of chemicallyaltered oligonucleotides in the final product.

Because these chemical alterations represent relatively subtle changes,synthetic oligonucleotides are nevertheless suitable for many purposes.For example, the chemical alterations in synthetic oligonucleotides donot significantly interfere with hybridization, enabling theseoligonucleotides to function effectively as DNA probes in mostdiagnostic applications. Similarly, in site directed mutagenesis, whereentire colonies or plaques are derived from a single oligonucleotideincorporation, a biological screen is inherently provided for thedesired mutation event.

However, certain applications require that a higher integrity product beemployed to achieve optimal results. Chemical alteration typicallybecomes a problem where the oligonucleotides are used in an applicationwhich requires or induces recognition of the oligonucleotide by abiologically active substance, most typically an enzyme. For example,oligonucleotide integrity becomes an issue in gene assembly wherepolymerase and ligase recognition of the oligonucleotide is required forcloning. This problem is particularly acute in the assembly of longergenes, because the opportunity for error to arise from chemicalalteration increases proportionately with the length of the gene.

The issue of oligonucleotide integrity is even more problematic in atherapeutic application where the therapeutic oligonucleotide mustinteract with a variety of biologically active components in thepatient's body. Further, any degree of incomplete deprotection orchemical modification in the therapeutic oligonucleotide product mighttrigger an immune response, or worse, impart a cytotoxic effect to thepatient. It is therefore questionable whether synthetic oligonucleotidescan be effectively employed as therapeutic agents. This problem isfurther exacerbated by the fact that the exact nature of these subtlechanges or errors are difficult, if not impossible, to characterize bycurrent methods of chemical analysis. Mandecki et al, Biotechniques,9(1), 56-59 (1990).

Traditional cloning techniques provide an alternative method forproducing oligonucleotides. In cloning, the desired oligonucleotidesequence is inserted into an appropriate vector which is subsequentlyused to transform a host cell which, as it grows, amplifies the insertedoligonucleotide. The oligonucleotide can be synthesized in vitro andthen inserted into a vector which is amplified by growth, as disclosedin U.S. Pat. No. 4,293,652. The inserted oligonucleotide sequence, ifappropriately designed, can then be excised, for example by cutting witha restriction enzyme, and subsequently purified from extraneous plasmidDNA by standard techniques.

Oligonucleotides generated by cloning do not carry the danger ofchemical modification. As a result, these oligonucleotide productsappear as native, or "wild type", oligonucleotides which will, forexample, be recognized by enzymes. Cloning procedures, however, do nottend to be economical or amenable to large scale production ofoligonucleotides. Because the inserted oligonucleotide sequencerepresents only a small portion of the plasmid sequence, the weightyield of product DNA is very small compared with the weight yield of theplasmid. Even the insertion of tandem repeats of the desiredoligonucleotide sequence is likely to be of no avail in improving yield,because biological host cell systems typically excise tandem repeatsduring growth.

Target amplification, a technique which has been employed to improvesensitivity in probe diagnostics, provides yet another, albeit"untraditional", type of oligonucleotide synthesis. For example, U.S.Pat. Nos. 4,683,195 and 4,683,202 disclose a process known as polymerasechain reaction (PCR), wherein two oligonucleotide primers are employedsuch that the primers are complementary to the ends of differentportions on opposite strands of a section of the target sequence.Following hybridization of these primers to the target, extensionproducts complementary to the target sequence are formed in the presenceof DNA polymerase and an excess of nucleoside triphosphates. The primersare oriented so that DNA synthesis by the polymerase proceeds across andthrough the region between the primers. The hybridized extension productis then denatured from the target and the cycle repeated, with extensionproduct also acting as template for the formation of additionalextension product in subsequent cycles of amplification. Each successivecycle theoretically doubles the amount of nucleic acid synthesized inthe previous cycle, resulting in exponential accumulation of amplifiedproduct.

The PCR technology has been suggested for use as a method to achieve theproduction of large quantities of oligonucleotides, in European PatentApplication No. 359,545. PCR production of oligonucleotides is, however,still limited with respect to the quantity of final oligonucleotideproduct which can be generated by this method, because a stoichiometricamount of each synthetic oligonucleotide primer must be incorporatedinto each PCR extension product. (In reality, a large excess of theseprimers is required to drive the kinetics of the PCR reaction forward.)

Another drawback, most typically in therapeutic applications, lies inthe inability of PCR to completely eliminate the nucleic acid integrityproblems of organic synthesis, because the synthetic primers in factbecome a portion of the PCR-amplified oligonucleotide. As a result, theprimer-derived portion of the product will contain the same errors thatcompromise the quality of synthetic oligonucleotides. For this reason,PCR amplification products cannot be of significantly higher qualitythan than that of the starting oligonucleotide primers. Additionally,the Thermus aquaticus (Taq) DNA polymerase enzyme which is typicallyused in PCR amplifications is believed to add one or more nucleotidesduring amplification onto the 3'-ends of the products beyond theoligonucleotide primers. Denney et al, Amplifications, A forum for PCRusers, 4, 25-26 (March, 1990). It is also believed that Taq DNApolymerase can add bases onto the 3'-end of oligonucleotides insolution. Denney et al, ibid. This would result in a product which isnot sequence specific.

Other types of target amplification which have been developed fordiagnostic applications also warrant mention. International PatentApplication No. 89/02649 discloses a type of amplification proceduregenerally referred to as ligase chain reaction (LCR), whereinpresynthesized pairs of amplification probes which hybridizecontiguously to a section of the target sequence are ligated to form thecomplementary amplification product. As with PCR, the completedamplification product is separated from the target by heat denaturation,and the process repeated with both the target and amplification productacting as a template in subsequent cycles. The primary advantage of theLCR method over prior organic synthesis methods lies in its ability togenerate longer oligonucleotides from the shorter presynthesizedamplification probes. The final oligonucleotide product can besynthetic, wild type, or a combination of both, depending upon thecomposition of the presynthesized probes.

Yet another type of diagnostic amplification method, referred to ascatalytic hybridization amplification ("CHA"), is disclosed inInternational Patent Application No. 89/09284. In this method, multiplecopies of the complement of a portion of a target sequence are"generated" from the target-catalyzed cleavage of longer presynthesizedcomplementary probe sequences. Cleavage of the longer probes occurs onlywhere the target hybridizes to one of an excess of these complementarydetection probes in such a way that the target sequence catalyzesselective cleavage of only that complementary probe. Because the targetremains intact, it can be recycled through the reaction, enabling it toreact with more than one of the complementary detection probes, thusleading to a large amplification of signal through proliferation of theshorter cleaved pieces. CHA reactions, however, fail to provide anadvantage in the synthesis of oligonucleotide products, because longerpresynthesized oligonucleotides are simply being cleaved into smallerpieces.

It is an object of the present invention to provide a cost efficientmethod for producing large quantities of high quality oligonucleotides.

It is a further object of the present invention to provide a method forproducing large quantities of oligonucleotides that can be useful in adiagnostic setting.

SUMMARY OF THE INVENTION

The present invention uses a specially designed template sequence,otherwise referred to as a "substrate reagent", to catalyticallyassemble sequence specific oligonucleotide products from individualnucleoside triphosphate reagents in the presence of a polymerase and acutting agent. Because the process is catalytic with respect to thetemplate sequence, a virtually unlimited amount of product can begenerated from a single molecule of template. A cascade can be formedfrom a series of these substrate reagent templates, wherein the nucleicacid product from one substrate reagent template catalyzes a synthesisreaction using the next substrate reagent in the series. Where theprocess is initiated by the presence of a target nucleic acid sequence,the method of the present invention can be used as an amplificationmethod to improve sensitivity in a diagnostic setting. The catalyticsynthesis of oligonucleotides can conveniently be made to proceedisothermally, even where a cascade is employed.

The invention further provides a diagnostic kit comprising a nucleicacid template sequence precursor, an excess of deoxynucleosidetriphosphates, an agent for polymerization, and a nuclease, wherein saidtemplate sequence precursor has a cutting attenuation modification thatprevents cleavage of the template precursor during selective nucleasecleavage of an extension of the template sequence and wherein saidtemplate sequence precursor is complementary to a portion of a targetsequence.

The invention still further provides a diagnostic kit comprising a firstnucleic acid template sequence precursor, a second nucleic acid templatesequence precursor, a ligase and a nuclease, wherein one of said firstor second template sequence precursors has a cutting attenuationmodification that prevents cleavage of the template precursor duringselective nuclease cleavage of an extension of the template sequence andwherein both of said template sequence precursors are complementary tocontiguous portions of a target sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the elements of a substrate reagent of the presentinvention.

FIG. 2 demonstrates the in situ synthesis of an oligonucleotide sequencefrom a substrate reagent of the present invention.

FIG. 3 is an example of a "self-priming" method of target initiationscheme wherein the target acts as a ligation template for twocontiguously hybridizing substrate precursors.

FIG. 4 is an example of a "target-priming" method of target initiationscheme wherein the target acts as a ligation template for twocontiguously hybridizing substrate precursors.

FIG. 5 is an example of "target-priming" method of target initiationscheme wherein the target acts as a polymerization template for a singlepriming substrate precursors.

FIG. 6 is an illustration of a two level cascade using a series of twosubstrate reagents of the present invention.

FIG. 7 graphically illustrates the fold amplification versus cycles fora one, two, and three level cascade.

FIG. 8 demonstrates the undesired production of spurious cleavageproduct in a diagnostic setting from a self-primed substrate reagentwhich has folded back on itself.

FIG. 9 demonstrates the undesired production of spurious cleavageproduct in a diagnostic setting from a substrate reagent that isnonspecifically primed with carrier DNA.

FIG. 10 demonstrates the production of cleavage product from a two levelcascade using catalytic primers which contain a tailing sequence, wherethe tailing contains a remote recognition site.

FIG. 11 illustrates the oligonucleotide sequences and phosphatemodifications used in Example 2 to screen for potential cuttingattenuation modifications.

FIGS. 12A and 12B are the autoradiograms of polyacrylamide gels used toseparate the products generated in Example 2.

FIG. 13 illustrates the oligonucleotide sequences and cuttingattenuation modifications used in Example 3 to evaluate potentialinterference of the polymerase reaction by various cutting attenuationmodifications.

FIG. 14 shows the autoradiogram of polyacrylamide gels used to separatethe products generated in Example 3.

FIG. 15 illustrates the oligonucleotide sequences used to generate acleavage product from a primed substrate reagent, as described inExample 4.

FIG. 16 shows the autoradiogram of polyacrylamide gels used to separatethe products generated in Example 4.

FIG. 17 illustrates the oligonucleotide sequences used to demonstratecascade production of cleavage product, as described in Example 4.

FIG. 18 shows the autoradiogram of polyacrylamide gels used to separatethe products generated in Example 5.

FIG. 19 illustrates the oligonucleotide sequences used to generate acompleted substrate reagent from two substrate precursors in thepresence of ligase, using target as a template, as described in Example6.

FIG. 20 is the PhosphorImager™ graphic printout of the polyacrylamidegel used to separate the products generated in Example 6.

FIG. 21 illustrates the oligonucleotide sequences used to generate acompleted substrate reagent from a single substrate precursor in thepresence of an excess of dNTP's and polymerase, using target as atemplate, as described in Example 7.

FIG. 22 is the PhosphorImager™ graphic printout of the polyacrylamidegel used to separate the products generated in Example 7.

FIG. 23 illustrates the oligonucleotide sequences used to generate acleavage product from a target-initiated substrate reagent, as describedin Examples 8 and 9.

FIG. 24 is the PhosphorImager™ graphic printout of the polyacrylamidegel used to separate the products generated in Example 8.

FIG. 25 is the PhosphorImager™ graphic printout of the polyacrylamidegel used to separate the products generated in Example 9.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for the catalytic assembly ofsequence specific oligonucleotide products from a specially designedtemplate in the presence of a polymerizing agent, an excess of monomericnucleoside triphosphates, and a cutting agent. A substrate reagent isprovided in the form of a modified template sequence. A cuttingattenuation modification to the template sequence imparts resistance tocutting by a selected cutting agent, such as a restriction enzyme. Thecutting attenuation modification enables the substrate reagent to remainintact following selective cleavage to generate the desiredoligonucleotide. In this way, the substrate reagent can act as templatefor the in situ synthesis of the desired oligonucleotide over and overagain.

The catalytic process of the present invention enables the production ofan almost unlimited amount of oligonucleotide product from a singlesubstrate reagent template. The product produced from the substratereagent is of a native, or "wild type", quality because it is derivedfrom "native" nucleoside triphosphates (dNTP's) and a DNA polymerase.Moreover, a series of substrate reagents can be employed to form aseries of levels within a cascade to isothermally generate product. Inthe cascade, the product from the first level catalyzes the generationof product at the second level, and so forth. This results in anexponential accumulation of product, and is particularly useful in adiagnostic setting where the presence of target is used to initiate thecascade.

In order to more clearly understand the invention, it will be useful toset forth the definitions of certain terms that will be used herein:

Nucleic Acid Sequence or Oligonucleotide is a deoxyribonucleotide or aribonucleotide which may be modified with respect to: (1) the phosphatebackbone; (2) the nucleosides; and/or, (3) the ribose, or sugar moietyof the oligonucleotide. Nucleic Acid Sequences may contain labels orother attached moieties and can be interrupted by the presence of stillother moieties, as long as hybridization can occur.

Substrate Reagent is a nucleic acid sequence template which has acutting attenuation modification. When primed by the hybridization of acomplementary catalytic primer to form a partial duplex, the SubstrateReagent acts as a template for the in situ synthesis of an extendedsubstrate reagent. The Substrate Reagent may be pre-primed.

Cutting Attenuation Modification is a modification to the substratereagent which will cause a cutting agent to selectively cleave thedesired oligonucleotide cleavage product from an extended substratereagent without cutting the substrate reagent template.

Selective Cleavage means that substantially only one strand of a duplexnucleic acid sequence is cleaved, although cleavage of both strands mayoccur to some extent.

Cutting Attenuation Efficiency is a measure of the degree to which thecutting attenuation modification prevents cleavage of the substratereagent template during selective cleavage of the extended substratereagent. The Cutting Attenuation Efficiency represents the proportion ofmodified nucleic acid sequence which remains intact following contactwith a cutting agent through one cycle of extension and selectivecleavage of a substrate reagent.

Catalytic Primer as used herein refers to a nucleic acid sequence whichis complementary to a portion of a substrate reagent at or near the3'-end of the substrate reagent and which is capable of priming thesubstrate reagent by acting as a point of initiation for the synthesisof an extended substrate reagent using the unhybridized single-strandedremainder of the primed substrate reagent as a template for polymeraseextension.

Primed Substrate Reagent as used herein means a substrate reagent towhich a catalytic primer has hybridized.

Pre-primed means the catalytic primer is provided in the reactionmixture as a presynthesized reagent.

Extended Substrate Reagent is the duplex product which is generated fromthe extension of a primed substrate reagent or an activated substratereagent in the presence of an excess of nucleoside triphosphates and anagent for polymerization such as DNA polymerase. The Extended SubstrateReagent is extended substantially the entire length of the substratereagent.

Cleavage Product is the single-stranded polymerase extendedoligonucleotide product which is released by partial denaturation of aselectively cleaved extended substrate reagent.

Partial Denaturation refers to the denaturation of a portion of aselectively cleaved duplex. Partial Denaturation may, for example, referto the release of a cleavage product from an extended substrate reagentwhich has been selectively cleaved. Partial Denaturation may alsodescribe the denaturation of a portion of target sequence from asubstrate reagent-target duplex during target initiation.

Activated Substrate Reagent is the partial duplex product which remainsfollowing partial denaturation of a selectively cleaved extendedsubstrate reagent to release a cleavage product.

Partial Duplex may refer to either or both a primed substrate reagentand/or an activated substrate reagent.

Intervening Region is an optional portion of a substrate reagent whichlies between the priming region and the complementary region. TheIntervening Region may also be referred to as a Locking Region, becauseit can serve to "lock" the catalytic primer in place.

Cutting Agent is a chemically or biologically active substance, such asa restriction endonuclease, which selectively cleaves double-strandednucleic acid sequences. Typically, the Cutting Agent selectively cleavesextended substrate reagent (leaving the substrate reagent templatesequence intact) to generate cleavage product and an activated substratereagent. The Cutting Agent may also selectively cleave the targetportion of a substrate reagent-target duplex during target initiation.

Recognition Site as used herein means the specific sequence recognizedby an enzyme, such as a restriction endonuclease.

Enzyme Cleavage Site means the phosphodiester bond which is hydrolyzedby an enzyme, such as a restriction endonuclease.

Remote Cutting Restriction Endonuclease, or Remote Cutter, is arestriction endonuclease that cleaves double-stranded DNA at a siteoutside of the enzyme recognition site.

Restriction Site may refer to both the recognition site and the enzymecleavage site for a non-remote cutting restriction enzyme.

Cascade refers a series of substrate reagents wherein the cleavageproduct from one substrate reagent catalyzes, or primes, the nextsubstrate reagent in the series. The first substrate reagent in theseries must be pre-primed.

First Level refers to the first in a series of substrate reagents whichform a cascade.

Higher Level refers to any substrate reagent other than the first in aseries of substrate reagents which form a cascade.

Amplify or Amplification means to generate a greater quantity of finaloligonucleotide product than starting oligonucleotide, such as target,through the catalytic action of at least one primed or activatedsubstrate reagent.

Amplification Cascade as used herein refers to the situation where acascade is employed to achieve amplification of a startingoligonucleotide sequence. In an Amplification Cascade, the firstsubstrate reagent in the series is pre-primed through target initiation.

Target Initiation refers to target-induced in situ synthesis of asubstrate reagent.

Target Sequence is the nucleotide sequence being sought in a particularassay.

Presynthesized nucleic acid sequence as used herein means anoligonucleotide sequence which has been synthesized prior to being addedto a reaction mixture.

Complementary refers to sufficient complementarity to enablehybridization and/or extension to occur. Complete complementarity is notrequired.

Catalytic Production of Oliconucleotides

The nucleic acid substrate reagent is central to the present invention.Although the exact composition of the substrate reagent will vary,depending upon the intended use of the final oligonucleotide cleavageproduct, the substrate reagent nevertheless contains a minimum of tworegions. In most cases, the substrate reagent will contain at leastthree regions, as shown in FIG. 1. A first required region iscomplementary in sequence to the desired oligonucleotide product, and isreferred to as the "complementary region". A second required region ofthe substrate reagent is complementary to at least a substantial portionof a catalytic primer which primes synthesis of the desiredoligonucleotide sequence from a 3'-hydroxyl group using a polymerase anddNTP's, and is referred to as the "priming region". The complementaryregion and the priming region may be contiguous, or these regions may beseparated by a third region referred to as an intervening region.

The priming region of the substrate reagent will typically be pre-primedfor the synthesis of the desired oligonucleotide cleavage product. Forexample, the catalytic primer may be provided as a separatepresynthesized reagent. In order to facilitate hybridization of thecatalytic primer to the substrate reagent, both the catalytic primer andthe substrate reagent may be introduced as a composite reagent with thecatalytic primer already annealed to the priming region. Where thelatter approach is taken, it may be convenient for the catalytic primerto be covalently attached to the priming region of the substrate reagenttemplate, such as through a linker arm as shown in FIG. 1. It is mostpractical, and therefore preferred, that the linker arm be a nucleicacid sequence.

The substrate reagent will contain a cutting attenuation modificationwhich allows for the selective cleavage of the synthesizedoligonucleotide product from the extended substrate reagent. Because thecutting agent selectively cleaves only the polymerase extended strand ofthe extended substrate reagent, the substrate reagent template remainssubstantially intact. Preferably, the cutting attenuation modificationwill impart complete resistance to cutting. It is also acceptable,however, for the modification to attenuate cutting to a substantial, butless than complete, degree. Cutting attenuation efficiencies as low as50% may function adequately in the method of the present invention,depending upon the particular substrate reagent design and desired enduse of the oligonucleotide cleavage product.

It will be appreciated that loss of substrate reagent, due to cleavageof the substrate reagent from cutting attenuation efficiencies of lessthan 100%, will increase with each cycle, thus having a substantialeffect on the overall efficiency of the system. For example, a cuttingattenuation modification with 50% cutting attenuation efficiency willresult in a loss of approximately 50% of the total amount of primedsubstrate reagent after the first cycle, 75% after the second cycle, andso forth. The loss of substrate reagent from lower cutting attenuationefficiencies is less critical where lesser amounts of finaloligonucleotide cleavage product are desired, because fewer cycles ofoligonucleotide production will be required to achieve the desired endresult. The negative impact of low cutting efficiencies may also beovercome to some degree where the cascade format of the presentinvention is employed, as later described.

The cutting attenuation modification is important, because it enablesthe substrate reagent to be used over and over again, imparting thedesired catalytic behavior to the system. For this reason, it ispreferred that the cutting attenuation modification provide at least 60%cutting attenuation efficiency. It is more preferred that cuttingattenuation efficiency be at least 90%. Most preferably, the cuttingattenuation modification will provide a cutting attenuation efficiencyof at least 95%. It is also preferred that the cutting attenuationmodification not interfere significantly with polymerase activity acrossthe substrate reagent template. It may be necessary to screen potentialcutting attenuation modifications, as taught in the examples whichfollow, to identify an optimal cutting attenuation modification for aparticular system.

As noted, a cutting agent is employed to effect the release of thesynthesized oligonucleotide product from the extended substrate reagent.Like the catalytic primer, this agent can either be attached to thesubstrate reagent, or it can be provided separately. The cutting agentwill generally be a chemically or biologically active substance. What isimportant in the selection of a cutting agent is that the cutting agentselectively cleave the desired oligonucleotide cleavage product from theextended substrate reagent while leaving the substrate reagent templatesequence substantially intact. It will therefore be appreciated that thecutting attenuation modification and the cutting agent operate intandem. A preferred cutting agent/cutting attenuation modificationcombination is a restriction endonuclease with a corresponding chemicalmodification in the substrate reagent backbone. In this case, thebackbone modification renders the substrate reagent resistant torestriction endonuclease cleavage.

In addition to the preferred restriction enzyme cutting agent, sequencespecific mammalian endonucleases, such as recombinases, can also act ascutting agents according to the present invention. These endonucleasesare less well characterized than restriction enzymes, but can provideimportant advantages for the in vivo generation of oligonucleotidecleavage products.

It is further possible to use chemical means of cleavage as the cuttingagent. For example, certain chelator-metal complexes have been tetheredto oligonucleotide sequences on either the 3'- or 5'-ends to effectcleavage of a corresponding complementary nucleic acid strand. Thesechelator-metal complexes include: EDTA-Fe (Boutorin et al, FEBS Lett.,172, 43-46 (1984)); phenanthroline-Cu (Chen et al, J. Amer. Chem. Soc.,110, 6570-6572 (1988)); and porphyrin-Fe/Co/Mn (Le Doan et al,Biochemistry, 25, 6736-6739 (1986)). Cleavage of the complementarynucleic acids is induced through the production of the OH.sup.. radicalswhich result from a localized concentration of hydrogen peroxideproduced by dismutation of superoxide anion O₂.sup..-. The superoxideanion is produced spontaneously as a result of electron transfer fromthe metal to molecular oxygen.

These chelator-metal complexes are known to cause the cleavage ofnucleic acids on the opposite strand of the tethered strand withoutattacking the tethered strand to which the chelator-metal complex isattached. The resistance of the tethered strand to the cutting action ofthe chelator-metal complexes is probably due to steric constraintsimposed by the tether. In this case, the substrate reagent of thepresent invention would be labeled with the chelator-metal complex,which would then cut the upper strand of the extended substrate reagent.The cutting attenuation modification in this case is the tether, whichnot only attaches the complex to the substrate reagent, but which alsocauses the steric constraint(s).

The design of the substrate reagent is critical to its efficient use inthe method of the present invention. Where presynthesized substrateprecursors are used to form the substrate reagent, as in the case oftarget initiation described below, the design of these substrateprecursors is also critical. Once designed, synthesis of the substratereagent and/or substrate precursors can be performed according to anyknown technique. Many such techniques, including those described in thebackground of this patent application, are known to those skilled in theart. It is preferred that the integrity of the substrate reagent be asclose to a naturally occurring oligonucleotide sequence as possible inorder to facilitate recognition by a biologically active polymerase.

In designing the substrate reagent, the complementary portion of thesubstrate reagent is selected to be complementary to the desiredoligonucleotide product. Theoretically, the method of the presentinvention can be used to generate cleavage products ranging in size from1 to several thousand nucleotides in length. Preferably, where anisothermal process is employed, the oligonucleotide cleavage productwill be no more than about 60 nucleotide bases in length. Morepreferably, the desired oligonucleotide cleavage product will also be atleast about 6 to 12 nucleotide bases long. A length of approximately 6bases or longer is generally required for oligonucleotides to functionas linkers for cloning or for primers in polymerase extension reactions.Oligonucleotides of approximately 12 bases or longer are required foruse as sequence specific probes for simple genomes such as E. coli. Theupper limit of approximately 60 nucleotide bases is established forisothermal processes, because the melting temperatures (T_(m)) of longeroligonucleotide products converge upon the same value at or about thispoint. Thus, oligonucleotides longer than about 60 bases cannot bedifferentiated or selectively denatured by temperature adjustment(s).For example, the T_(m) of a 60-mer oligonucleotide may be identical tothat of a 100-mer oligonucleotide, making it difficult, if notimpossible, to identify a temperature which will allow for the selectivepartial denaturation of a desired 60-mer oligonucleotide cleavageproduct while leaving a 100-mer catalytic primer hybridized to thesubstrate reagent template.

Within these limitations, the exact length of the complementary region,and corresponding cleavage product will be determined by the desired enduse of the oligonucleotide cleavage product. Antisense oligonucleotides,for example, must be effective at physiological temperatures, and aretypically about 15 to 25 nucleotides long. Generally, longer antisenseoligonucleotides within this range are desirable, because they have alower probability of occurring by chance in large genomes. For example,a 17-mer oligonucleotide should be unique to a mammalian genome. On theother hand, if an antisense oligonucleotide is too long (i.e.,substantially longer than 25 nucleotides), it may hybridizenonspecifically to longer non-target sequences. This type of nonspecifichybridization is unavoidable, because the physiological body temperatureof a patient cannot be adjusted to increase stringency.

Nonionic antisense oligonucleotides have a slightly higher T_(m), due tothe lack of phosphate charge repulsion, and will typically be selectedat the shorter end of the 15 to 25 nucleotide range than will unalteredionic oligonucleotides. Phosphorothioates and phosphorodithioates, onthe other hand, have a slightly lower T_(m). These oligonucleotides maybe adjusted to be as much as 25 to 30 nucleotides long. It is possibleto incorporate phosphorothioate linkages into DNA by polymeraseextension in the presence of dNTPαS (2'-deoxynucleoside5'-o-(1-thiotriphosphate)) and a suitable template. Eckstein et al, Nuc.Acids Res., 13(24), 8749-8764 (1985). Many restriction endonucleases areable to cleave this modified phosphate internucleotide bond, and ittherefore should be possible to produce phosphorothioate-containingcleavage products using the present invention. Furthermore, since onlythe S_(p) optical isomer serves as a substrate for DNA polymerases, theresulting cleavage products will be optically pure.

The cleavage product of the present invention may also be used as anoligonucleotide probe or primer. In this case, the optimal size of thecleavage product will vary with respect to the intended assayconfiguration. Because the stringencies of an in vitro probe applicationcan be adjusted, the range of potential probe sizes is far greater thanthat allowable for antisense oligonucleotides. Nevertheless, the probeor primer size should be adjusted to be unique with respect to the sizeof the genome in the analyte. For example, PCR probes are generally 15to 35 base pairs in length, while LCR probes are typically 8 to 35 basepairs in length.

The priming and intervening regions of the substrate reagent can be anyoligonucleotide sequence that is convenient for practical purposes. Forexample, it may be convenient for the priming region to be complementaryto a readily available supply of oligonucleotide sequences, such asdiagnostic probes or primers. It is further preferable that the primingregion not be complementary to the complementary and/or interveningregion(s) of the substrate reagent, as this can result in the substratereagent folding back on itself. The catalytic primer will preferablyhave a higher T_(m) than the oligonucleotide cleavage product, as thiswill aid in keeping the catalytic primer from melting off duringdenaturation to effect release of the cleavage product. This usuallymeans that the priming region will be at least as long as theoligonucleotide cleavage product, particularly where the substratereagent does not have a locking region. More preferably, the catalyticprimer will be 1-5 bases longer than the cleavage product. Typically, a8- to 9-mer oligonucleotide is sufficient to achieve priming.

Unlike the complementary region, which must be substantially the samelength as its complementary cleavage product, it is not necessary thatthe priming region be substantially the same length as the complementarycatalytic primer. For example, the 5'-end of the catalytic primer mayextend beyond the 3'-end of the (priming region of) the substratereagent, providing a "tailing sequence" on the catalytic primer. Infact, it may be beneficial in some cases to use the tailing sequence asa template for polymerase extension such that the substrate reagent actsas a primer for extension opposite the tailing sequence. It is alsopossible for the 3'-end of the substrate reagent to extend beyond the5'-end of the catalytic primer, providing an optional "capping region"on the substrate reagent, as more fully described later.

Where an intervening region is part of the substrate reagent, greaterflexibility is available in the selection of a catalytic primer. With anintervening region, the catalytic primer can actually be shorter thanthe cleavage product, because the added length provided by polymeraseextension opposite the intervening region may be used to effectively"lock" this shorter catalytic primer onto the substrate reagent at atemperature which still allows for denaturation of the cleavedoligonucleotide product. Essentially, the catalytic primer will be anoligonucleotide having a sufficiently high T_(m) to enable anappreciable degree of hybridization to the priming region of thesubstrate reagent. In this case, the polymerization reaction can beginquickly enough to lock the catalytic primer onto the substrate reagent.With an intervening region, the catalytic primer can be as much asseveral bases shorter than the cleavage product. In any event, the totallength of the priming region plus intervening region will preferably beat least as long as the cleavage product.

The cutting attenuation modification may be located within any of theregions of the substrate reagent depending upon the particular design ofthe system. The precise location of the cutting attenuation modificationwill vary with respect to the exact cutting attenuation modification andcutting agent selected. Optimal positioning of the modification will beapparent to one of ordinary skill in the art through minimalexperimentation following the teachings of the present invention.Incorporation of the selected cutting attenuation modification into thesubstrate reagent will generally be made through the use of anappropriately modified nucleotide, ribose, or phosphate at a preselectedpoint during synthesis of the substrate reagent.

The substrate reagent is provided in a reaction mixture with thecorresponding catalytic primer and cutting agent, an agent forpolymerization, and an excess of dNTP's. All of the reagents may beprovided initially. Sequential addition is unnecessary. FIG. 2demonstrates the synthesis of an oligonucleotide cleavage product from apre-primed substrate reagent which does not contain the optional linkerarm of the present invention. The pre-primed substrate reagent shown inFIG. 2 contains an intervening region between the priming region and thecomplementary region. The cutting attenuation modification of thesubstrate reagent in FIG. 2 is provided in the form of a backbonemodification located within the complementary region of the substratereagent, with the cutting agent being provided as a correspondingrestriction endonuclease. The primed substrate reagent is contacted witha DNA polymerase and an excess of dNTP's, which results in extensionthrough the intervening sequence and the complementary region to createan extended substrate reagent. Formation of the extension productnecessarily generates a restriction endonuclease recognition site on theextended upper strand near the cutting attenuation modification site.

As shown in FIG. 2, contacting the extended substrate reagent with theappropriate corresponding restriction endonuclease results in cleavageof the internucleotide bond which covalently binds the desiredoligonucleotide product (CP) to the remainder of the extended portion ofthe extended substrate reagent. The corresponding cutting attenuationmodification on the substrate reagent template, however, impedesconcurrent cutting of the complementary template strand. The action ofthe cutting agent leaves the desired oligonucleotide product bound tothe remainder of the extended substrate reagent only by hydrogen bondingwith the strand. The reaction mixture can therefore be held at atemperature at or near the T_(m) of the desired oligonucleotide cleavageproduct to effect release of this cleavage product.

The action of the cutting agent and subsequent denaturation of cleavageproduct from the substrate reagent template also generates an activatedsubstrate reagent which can again be extended by polymerase action withan excess of dNTP's to regenerate the extended substrate reagent foranother cycle of oligonucleotide synthesis. The activated substratereagent differs from the primed substrate reagent in the number ofindividual nucleoside bases which must be added by extension to generateextended substrate reagent. The activated substrate reagent is referredto as "activated", rather than "primed", because it requires addition ofonly the bases that will make up the resulting cleavage product in orderto generate another copy of cleavage product. If there is no interveningregion between the priming region and the complementary region, theprimed substrate reagent and activated substrate reagent will beidentical.

In the presence of a polymerase and dNTP's, the activated substratereagent will regenerate the extended substrate reagent, leading to yetanother copy of the desired oligonucleotide cleavage product (CP fromFIG. 2) following cleavage by the selected cutting agent. Importantly,all of these processes can occur rapidly in the same reaction mixturewithout the need for thermocycling or the addition of fresh reagents.The net result of this process is the repetitive catalytic conversion ofdNTP's into the desired oligonucleotide cleavage product underisothermal conditions. (It should be noted that oligonucleotidesproduced by this process will typically be phosphorylated on their5'-ends, depending upon the particular restriction enzyme used as thecutting agent.)

One of the advantages of the method of the present invention is therelative cost savings achievable by generating oligonucleotide productsby this enzymatic means as compared with conventional organic synthesismethods. The cost savings are of particular benefit in the area ofoligonucleotide therapy, where gram quantities of material are projectedto be required for a single therapeutic dose. Although the commercialcost of the dNTP reagents required by the present method can be moreexpensive than the amidite reagents required by traditional organicsynthesis methodologies, this higher cost is more than offset when therelative efficiency of the amidite technologies is factored into thecomparison. Specifically, the phosphoramidite synthesis technologiesrequire a large excess of non-reusable reagents to insure highcondensation efficiencies, whereas nearly all of the more expensivedNTP's can be converted to product using the present method, making theoverall cost of nucleoside reagents for the method of the presentinvention considerably less than that of conventional methodologies.Moreover, a considerable portion of the expense of the dNTP reagentslies in the fact that these reagents are commercially available only asseparate dATP, dTTP, dCTP, and dGTP reagents. The present method,however, employs a mixture of these dNTP's. Thus, a commercial source ofa combined reagent containing all four dNTP's could reduce the cost ofreagents for the method of the present invention by as much as an orderof magnitude, because the cost of separating the dNTP's could beeliminated.

It is possible to use a mixture of all four dNTP's in the presentmethod, because the template provided by the substrate reagent performsthe task of selecting the appropriate dNTP from the dNTP pool to achievethe desired sequence of the finished oligonucleotide cleavage product.With current organic synthesis methods, this selection must be donemanually or by programming an automated sequenator, requiring thatseparate reagents be added in a step-by-step manner to achieve thedesired end product. This enables the method of the present invention togenerate the desired large quantities of oligonucleotide product withmuch greater speed than conventional methodologies.

Just as important as the cost and output advantages achievable by theenzymatic method of the present invention, is the high integrity of theoligonucleotide product that is produced in this manner. Therapeuticoligonucleotides generated enzymatically according to the presentinvention are of native quality and are, therefore, more likely to besuitable for administration to a patient than are the syntheticoligonucleotides derived from traditional organic synthesis techniques.The preference for wild type products lies in the lower probability ofthese products to induce immunogenicity and cytotoxicity reactions.Also, while racemic mixtures of optically active products, such asphosphorothioates, are formed according to traditional synthesisprocedures, it is possible to obtain optically pure preparations ofthese modified oligonucleotide products for therapeutic use by themethod of the present invention, as noted below.

Although cleavage products of wild type DNA (containing phosphate bonds)are acceptable and desired for most applications, it is also possible toincorporate modified nucleotides into the cleavage product usingmodified nucleoside triphosphates, provided these modified dNTP's do notinterfere with polymerase extension. For example, biotinylatednucleosides and/or nucleosides containing amine groups can beincorporated into DNA by polymerase using the desired modified dNTP(s).Ward et al, Proc. Natl. Acad. Sci. USA, 78(11), 6633-6637 (1981). Themodification(s) can be made to the final oligonucleotide product in thismanner using a dNTP pool wherein one, two, three, or all four of thedATP, dTTP, dCTP, and dGTP reagents are modified. Phosphorothioateinternucleotide bonds have also been created using polymerase anddNTPαS. Eckstein et al, ibid. In this case, only one of the two possiblediastereomers serves as a substrate for polymerase. It is thereforepossible to do stereoselective synthesis using the method of the presentinvention. For example, chemical synthesis of a 20-mer oligonucleotidecontaining phosphorothioate linkages, using racemic starting materials,will yield over 1,000,000 isomers. On the other hand, the method of thepresent invention will yield only one optically pure isomer.

Modified oligonucleotides may be preferred in some antisenseapplications. It may also be possible to generate, through theincorporation of modified nucleosides, an oligonucleotide cleavageproduct that can itself serve as a substrate reagent. In other words,the modified nucleosides could be used to incorporate a cuttingattenuation modification into the cleavage product. In the presence ofan appropriate catalytic primer, this cleavage product could then serveas a substrate reagent to generate its own cleavage product.

In vivo Production of Oligonucleotides

The catalytic method of the present invention is also adaptable for thein vivo, or intracellular, production of oligonucleotides from a primedsubstrate reagent. This in vivo reaction has potential for enabling theproduction of antisense oligonucleotides directly in the cells of thepatient or directly in bacterial cells residing within the patient. Inorder to achieve this type of result, the appropriate primed oractivated substrate reagent could be provided directly to a patient,such as in the form of an injectable therapeutic. Once inside the cellswithin the patient's body, the substrate reagent could thencatalytically generate the desired antisense oligonucleotide. Thecatalytic reaction would, of course have to occur isothermally at thepatient's body temperature.

Because only a catalytic amount of the substrate reagent is required togenerate a correspondingly high level of antisense oligonucleotide, thedose required for therapeutic action could be reduced significantly(e.g., from gram to milligram quantities). Not only would this reducethe cost of antisense therapy to a practical level, but it would alsoreduce the potential for immunogenicity reactions which are more likelyto result from the introduction of large amounts of a foreigntherapeutic into a patient. In other words, the required "large amount"of therapeutic cleavage product would be formed in the cell and would,therefore, necessarily be of native quality. There is also a measure ofconvenience and patient comfort achievable from the administration ofmilligram amounts of therapeutic through a single injection, rather thangram amounts through intravenous infusion.

The method of the present invention is particularly well suited for thisin vivo application, because endogenous polymerases anddeoxyribonucleoside triphosphates are available in the desired targetcell to support the intra-cellular synthesis of the desired antisenseoligonucleotide products. Target cells for the substrate reagenttherapeutic may include normal cells in the patient, as well as abnormalcells (e.g., virally infected) or bacterial cells which have invaded thepatient's body. Many of these cells also carry endogenous sequencespecific endonucleases which would be available to serve as cuttingagent(s) for appropriately designed substrate reagents. For example,most bacterial strains carry restriction endonucleases which could beused as the cutting agent. In this case, intracellular production of anantisense oligonucleotide could produce a desired cytotoxic effect, thushaving potential applicability as an antimicrobial therapeutic.

It is even conceivable that the presence of a particular target nucleicacid sequence in a cell could serve to assemble substrate precursorsinto an activated or primed substrate reagent. This "target-initiated"substrate reagent could produce multiple copies of, for example, anantisense oligonucleotide that imparts a desired cytotoxic effect. Inthis case, cells containing a particular target sequence such as thehuman immunodeficiency virus could be selectively destroyed by cytotoxicantisense oligonucleotides produced as a result of the target initiationphenomenon more fully described below.

Although mammalian cell lines are less well characterized than microbialcell lines, the former are also known to carry sequence specificendonucleases that are required for recombination events. For example,"stuffer" sequences located between a 7-mer and a 9-mer of definedsequence in the RSS sequence of immunoglobulin genes and in T-cellreceptor gene rearrangements have been observed to recombine in certainlymphoid cells. The "stuffer" region can be of any sequence, and couldtherefore serve as the cleavage product according to the presentinvention. ICSU Short Reports; Advances in Gene Technology, Proceedingsof the 1990 Miami Biotechnology Winter Symposia, 10, 21 (1990). Theserecombination events require double-strand cleavage by a recombinase atthe sites which are flanked by the sequence specific 7-mer and 9-mer. Inan in vivo application of the present invention, this same recombinasecould serve as the cutting agent for a substrate reagent therapeutic,provided the primed substrate reagent also contained two cuttingattenuation modifications at the junctions of the stuffer sequence toprevent cutting of the substrate reagent template. In cases where aparticular cell does not carry a sequence specific endonuclease, thecutting agent could be tethered to the primed substrate reagent or tothe appropriate substrate precursors from which it is derived.

Taraet Initiation

The catalytic oligonucleotide production method of the present inventioncan be initiated by the presence of a target. The primary advantage oftarget initiation is to enable oligonucleotide synthesis to be used fordiagnostic purposes as a method for amplification in response to targetsequence in a patient test sample. Target initiation can, however,provide other significant benefits, such as in the in vivo applicationsdiscussed above.

Target initiation requires that the target induce, or trigger, the insitu synthesis of a substrate reagent. This target-initiated substratereagent can be single-stranded or it can be a partial duplex in the formof either a primed substrate reagent or an activated substrate reagent.If the in situ-synthesized substrate reagent is single-stranded, acatalytic primer must be provided as an additional reagent. It istherefore preferred that the initiation process result in a substratereagent which is a partial duplex. Initiation can take place in any oneof a number of ways. Initiation methods can be isothermal, or they mayrequire thermocycling. There are advantages to both types of methods, aswill be discussed. In any event, it is preferred that the targetsequence initiate amplification by acting as a template to catalyze thein situ synthesis of a substrate reagent. In this case, it is convenientto use presynthesized nucleic acid sequences, referred to as substrateprecursor(s), to effect initiation.

The target sequence can act as a "ligation template" for the contiguoushybridization and subsequent ligation of two or more substrateprecursors to form the complete substrate reagent. It will generally bepreferred to use two substrate precursors in ligation template targetinitiation. In any event, the ligation template method of targetinitiation may be "self-priming" or "target-priming", as describedbelow, either of which may be preferred, depending upon whetherthermocycling is viewed as a disadvantage in a particular application oftarget initiation.

Where the self-priming embodiment is used, one of the substrateprecursors is synthesized so that it provides the catalytic primer forthe completed substrate reagent. For example, in the self-priming methodof ligation template target initiation shown in FIG. 3, one of thesubstrate precursors SP₁ contains a sequence which is complementary to aportion of the target sequence and an additional non-complementary(priming region) sequence which is hydrogen bonded to the catalyticprimer for the substrate reagent. The other substrate precursor SP₂ iscomplementary to a contiguous portion of the target sequence next to thenon-priming end of SP₁. The cutting attenuation modification ispreferably located on one of the substrate precursors at a positionselected to achieve the appropriate cutting by the cutting agent in theeventual oligonucleotide synthesis scheme.

Where a target is double-stranded, the target-containing sample mustfirst be denatured before it can anneal with the substrate precursors.Denaturation may take place in the presence of an excess of thesubstrate precursors, with annealing of the substrate precursors alsotaking place at a temperature dictated by the lower T_(m) of the twosubstrate precursors. Once annealed, the two contiguously hybridizedsubstrate precursors can be ligated to form a self-primed substratereagent. In this type of target initiation, a separate heating step isrequired to release the completed substrate reagent. It is important toprovide a large excess of the substrate precursors, as this not onlydrives the reaction forward, but also provides an excess of catalyticprimer provided by the non-complementary region of SP₁, so that ifcatalytic primer is melted off during denaturation, the substratereagent will quickly be re-primed from the excess of reagents provided.This process has the advantage that it can be repeated with thermalcycling to generate additional copies of the substrate reagent, as shownin FIG. 3.

In a target-priming type of ligation template target initiation, thetarget not only acts as a ligation template for the substrateprecursors, but also provides the catalytic primer necessary to form theprimed or activated substrate reagent. The target-primed initiationembodiment offers the advantage that once sample target DNA isdenatured, the remainder of the initiation process is isothermal. Thedisadvantage to this method is that only one copy of primed substratereagent is produced from each target sequence. Unlike the self-primingembodiment, SP₁ does not carry a separate catalytic primer region, asshown in FIG. 4.

In both the self-primed and target-primed ligation template initiationembodiment, the substrate precursors are preferably designed so that aligation event is required to generate a complete restriction site. Thecutting attenuation modification, positioned on SP₁ and/or SP₂, preventscutting of the assembled substrate reagent. In the target-primedligation method of target initiation shown in FIG. 4, activatedsubstrate reagent is formed by selective cleavage of the target strand,through the action of a cutting agent such as a restrictionendonuclease. This cutting agent is preferably the same cutting agentused to selectively cleave extended substrate reagent in the ensuingoligonucleotide production method.

In the case of target-primed initiation, it is preferred that the intactsubstrate reagent be released isothermally from the cleaved targetthrough partial denaturation. This result can be achieved in thetarget-primed embodiment through initial temperature adjustment of theinitiation reaction so that only that portion of the cleaved targetwhich is hybridized to the complementary region of the completedsubstrate reagent denatures from the substrate reagent, releasing anactivated substrate reagent. Isothermal denaturation avoids the need forthe separate heating step required to effect release of the assembledsubstrate reagent in the self-priming method. Although it is importantthat the remainder of the target, which primes the activated substratereagent, remain hybridized during denaturation of the cleaved targetportion from the complementary region, this result can also be easilyachieved by adjusting the T_(m) of this portion of the partial duplexthrough the use of a longer substrate precursor SP₁.

It is more preferred, however, to initiate amplification by using thetarget sequence as a polymerization template for a single substrateprecursor which primes extension opposite the target. In thepolymerization template method of target initiation, polymeraseextension occurs in the presence of the priming substrate reagent, anexcess of deoxynucleoside triphosphates, and an agent forpolymerization, as shown in FIG. 5. The polymerization template methodis particularly beneficial, because all of the necessary reagents,except for the substrate precursor, may be employed in the catalyticmethod for oligonucleotide generation which follows target initiation.

In the polymerization template target initiation scheme shown in FIG. 5,a single substrate precursor SP₁ is provided with a cutting attenuationmodification. This single substrate precursor is designed to hybridizeat or near a naturally occurring restriction site in the targetsequence. In this way, catalytic primer extension through the naturallyoccurring restriction site in the target creates a complementary site inthe substrate reagent which, but for the positioning of the cuttingattenuation modification on the substrate precursor primer, would be arestriction enzyme recognition site. The duplex thus formed between theextended modified catalytic primer and the target can then be contactedwith a cutting agent to selectively cleave the target, releasing anactivated substrate reagent. Just as in target-primed ligation templateinitiation, it is possible to achieve isothermal denaturation of theportion of the target not responsible for priming by initial temperatureselection for the reaction. This form of polymerization template targetinitiation is also target-primed, or in this case "target-activated",because cleavage of the target strand occurs at the same cutting sitewhich is preferably used in the subsequent catalytic oligonucleotideproduction method.

Still other variations of target initiation will be apparent to thoseskilled in the art, based upon the teachings of the present invention.These other methods may include additional ligation template initiationschemes as well as polymerization template initiation schemes. It willbe appreciated that "hybrid" methods employing both ligation templateand polymerization template schemes may provide additional advantages.

Cascade

The applicability of the catalytic oligonucleotide generation method ofthe present invention can be further broadened if a cascade of substratereagents is used. Although a single substrate reagent is sufficient andpreferred for traditional purposes of oligonucleotide synthesis, aseries of substrate reagents can be employed as a cascade, such that theproduct from the first level of the cascade catalyzes the generation ofproduct at the second level, and so forth, thereby dramaticallyincreasing the output of final oligonucleotide product, as shown in FIG.6. This cascade is of particular value in diagnostic applications wheretarget is present in extremely low quantities. (A single substratereagent may offer sufficient sensitivity in certain diagnosticsituations where the amount of target in a test sample is relativelyhigh.) In a typical diagnostic application, the target, although presentin minute quantity, is able to initiate the cascade, which then rapidlygenerates an exponentially greater quantity of final oligonucleotideproduct which can be measured in a traditional type of detection system.The amount of final oligonucleotide product can then be correlated backto determine the amount of original starting target oligonucleotide.

In employing a cascade, the substrate reagents and corresponding cuttingagent(s) are selected in much the same manner as for a one substratereagent reaction. Importantly, the first substrate reagent in the seriesmust be pre-primed or pre-activated. Where an amplification cascade isemployed in a diagnostic setting, target initiation will be required toproduce the first substrate reagent, thereby setting the cascade inmotion. The composition of this first substrate reagent will thereforebe dictated by the initiation scheme which has been selected. At allother levels of the amplification cascade, the substrate reagent ispresented as a complete, but unprimed, presynthesized reagent. Eachsubstrate reagent will typically be designed in the order of itsappearance in the cascade, since the cleavage product from the firstlevel must be complementary to the priming region of the second levelsubstrate reagent, and so forth. It is preferred to use the same cuttingagent and same cutting attenuation modification in the substrate reagentat all levels of the cascade in order to minimize the number of reagentsrequired to run the reaction.

It has unexpectedly been found that, like the single substrate reagentreaction, the cascade can proceed isothermally. In other words, thecleavage product from one level of the cascade can be made to denaturefrom its substrate reagent template and subsequently hybridize to thepriming region of the next substrate reagent for a sufficient length oftime to prime the in situ synthesis of oligonucleotide product at thisnew level of the cascade without a change in temperature. This isunexpected, because the T_(m) for the oligonucleotide cleavage productbound to its complementary substrate reagent is substantially identical,to the T_(m) for the same oligonucleotide product acting as catalyticprimer by hybridizing to the priming region of the next substratereagent in the cascade. (In fact, in the case where a tailing sequenceis incorporated into cleavage product/catalytic primer, the T_(m) forthe priming reaction will be lower.)

It is believed that the oligonucleotide cleavage product hybridizes tothe next level substrate reagent just long enough to allow forpolymerase extension to produce a longer oligonucleotide product with ahigher T_(m). This produces the net effect of "locking" theoligonucleotide onto the new substrate reagent so that it can no longerhybridize to the complementary portion of the previous substratereagent. The use of an intervening region is therefore particularlypreferred in a cascade, because it provides for the generation of anactivated substrate reagent with a higher T_(m) than the primedsubstrate reagent, making the activated substrate reagent more stablethan the primed substrate reagent.

It is imperative in a diagnostic setting that the substrate reagent(s)at higher levels of the cascade not be pre-primed, as generation ofcleavage product at each level must be triggered only by the presence oftarget. In the first level of a diagnostic amplification cascade,generation of cleavage product is triggered by target initiation whichprovides the first substrate reagent. Cleavage of the extended substratereagent not only generates catalytic primer for the next level of thecascade, but, as in the case of a single substrate reagent reaction, italso releases an activated substrate reagent to again act as a templatefor the in situ generation of another copy of extended substrate reagentat the current level. Once primed in the first instance by binding of acomplementary catalytic primer, the substrate reagent at higher levelsof the cascade need not be primed again in order to act as template forthe formation of additional extended substrate reagent in subsequentcycles of in situ synthesis at the same level of the amplificationcascade.

As each level beyond the first level of the cascade cycles, the amountof cleavage product generated at these higher levels grows exponentiallyas new primed substrate reagents are continually added to the partialduplex pool of recycled activated substrate reagents. This phenomenon isdemonstrated in FIG. 6. As shown in this two level amplificationcascade, the primed first substrate reagent pSR₁ acts as a template tocreate a first extended substrate reagent eSR₁. This extended substratereagent is then contacted with a cutting agent, in this case arestriction enzyme, which cuts only the upper strand, as illustrated, atpoint CS. The reaction is preferably run at a temperature such that thecleavage product CP₁ isothermally denatures from the cleaved extendedsubstrate reagent. Denaturation releases CP₁ to act as a catalyticprimer in the second level of the cascade, also releasing the activatedsubstrate reagent aSR₁ to be recycled in the first level of the cascade.Unlike higher levels, the amount of partial duplex in the first level ofthe cascade is "fixed", as all of the pSR₁ initially present (e.g., fromtarget initiation) is ultimately converted to aSR₁. Product which isgenerated from the fixed amount of target-initiated partial duplex (pSR₁or aSR₁, depending upon the particular initiation method) "accumulates"linearly at this first level. (There is no actual "accumulation" of CP₁,because virtually all of the CP₁ is consumed by the next level of thecascade.) This cycle of events will repeat itself m-1 times to produce mcopies of CP₁.

The released CP₁ serves as catalytic primer for the in situ synthesis ofa second extended substrate reagent (eSR₂) at the second level of theamplification cascade by priming SR₂. The eSR₂ is subsequently cleavedto release cleavage product CP₂ and the activated substrate reagentaSR₂. As described for the first level reaction, this second level willrepeat itself n-1 times to form a total of n copies of CP₂ from eachcopy of CP₁. In the case of the two level cascade shown in FIG. 6, CP₂will accumulate exponentially with respect to the starting targetnucleic acid sequence, because the supply of pSR₂ and aSR₂ iscontinuously increasing as a result of the continued production of CP₁from the first level of the cascade.

The yield of products in a particular cascade configuration follows anatural.geometric progression that can be calculated where certainassumptions and definitions are imposed upon the system. Theseassumptions are necessitated by the fact that cleavage product of thepresent invention is actually generated by way of two related, butslightly different, mechanisms. In the first mechanism, the cycle ofevents generating the cleavage product is initiated by hybridization ofa catalytic primer to a substrate reagent to form a primed substratereagent (pSR). The cycle involving the second mechanism begins with anactivated substrate reagent which has been generated from a previousfirst mechanism or second mechanism cycle. For purposes of defining thegeometric progression, these two process are assumed to proceed at thesame rate, with the production of one round of cleavage product fromeither of these processes being defined as one cycle. Following theseassumptions, the fold amplification (X) in a 1, 2, and 3 level cascadeafter "n" cycles can then be calculated according to the followingformula: ##EQU1##

Similarly, the fold amplification "X" in a cascade of any level "c"after a given number of cycles "n" may then be calculated according tothe following mathematical formula: ##EQU2##

Thus, after 100 cycling events, a one substrate reagent reaction willproduce 100 copies of cleavage product CP₁ for each copy of primedsubstrate reagent pSR₁. A two level cascade will produce 4,950 copies ofcleavage product CP₂ for each copy of first level primed substratereagent pSR₁, while a three level cascade will produce 161,700 copies ofcleavage product CP₃ for each copy of first level primed substratereagent pSR₁. This geometric accumulation of products is showngraphically for a one, two, and three level cascade in FIG. 7. A threelevel cascade will produce over one million copies of third levelcleavage product from each copy of primed first level substrate reagentafter only 183 cycles.

The total number of cleavage products N_(CP) produced in a reaction isthe product of the number of molecules of first level primed substratereagent N_(p) SR₁, and the fold amplification X_(c),n.

In diagnostic applications, the number of first level primed substratereagent pSR₁ molecules is directly proportional to the number of targetmolecules N_(T). In the "self-priming" embodiment of target initiation,one copy of pSR₁ is made from each target molecule each time the targetinitiation scheme is cycled. For the "target-priming" embodiment, asingle copy of pSR₁ is made from each molecule of target.

The basis for the success of the amplification cascade rests on thepriming of all higher level substrate reagent(s) by target-inducedcleavage product from the previous level. It is, however, possible forsome degree of non-target-induced priming of these unprimed substratereagents to occur from one of two possible phenomena. Production ofcleavage product by these processes is undesirable, because this"spurious" cleavage product introduces a background which can limit thesensitivity of the cascade diagnostic.

The first of these non-target induced priming phenomena occurs where thesubstrate reagent at higher levels exhibits a tendency to fold back onitself in such a way that it becomes self-priming. The potential forthis undesired type of self-priming is of particular concern in themajority of cascade formats employing restriction enzyme cutting agents,because most of these restriction enzymes normally generate5'-overhangs. In this case, the cleavage product from the previous levelsubstrate reagent will necessarily carry with it a significant portionof the restriction site. This creates certain design limitations fromthe fact that the priming end of the next higher level substrate reagentmust be complementary to the previous level cleavage product, and willtherefore also contain a significant portion of the restriction sitesequence. As a result, spurious cleavage product can be generated fromselective cleavage of the self-primed extended substrate reagent, asshown in FIG. 8.

The second phenomenon involves nonspecific priming of the unprimedhigher level substrate reagent(s) by the carrier DNA present in a testsample, as shown in FIG. 9. Carrier priming occurs where the 3'-end of acarrier DNA fragment partially hybridizes opposite the priming regionand/or intervening region of the substrate reagent long enough toinitiate polymerase extension. This typically leads to the generation ofspurious cleavage product from the resulting extended substrate reagent.Where a sufficient number of carrier DNA bases remain hybridized to thesubstrate reagent following selective cleavage and partial denaturation,the substrate reagent/carrier partial duplex may be stabilized againstdenaturation at the assay temperature of the cascade, creating aspurious activated substrate reagent. This problem is particularlyacute, because the spurious activated substrate reagent can now recycleto produce additional copies of spurious product.

The problem of non-target-induced priming of higher level substratereagents can be significantly overcome either by adding an optionalcapping region on the substrate reagent, or by providing a tailingsequence on the catalytic primer. In the latter case, the priming regionof each higher level substrate reagent in the amplification cascade willbe of a shorter length than the cleavage product (containing the tailingsequence) from the previous level of the cascade.

Where a capping region is used, it is placed at the the 3'-end of thesubstrate reagent contiguous to the priming region, such that the 3'-endof the substrate reagent extends beyond the 5'-end of the catalyticprimer. The function of the capping region is to prevent polymeraseextension of the 3'-end of the substrate reagent in the event that thesubstrate reagent folds back on itself. This desired end result ispreferably achieved by disrupting the 3'-end of the substrate reagent.For example, a dideoxynucleoside could be added to the 3'-end of thepriming region.

Where the 5'-end of the catalytic primer extends beyond the 3'-end ofthe substrate reagent, the "tailing sequence" of the extended portion ofthe catalytic primer provides a template which can be filled in by thesame polymerase and excess of dNTP's that are provided in the reactionmixture, as shown in FIG. 10. The use of a tailing sequence on thecatalytic primer is particularly advantageous when it is used incombination with a remote cutting restriction enzyme to effect cleavageof the extended substrate reagent. As shown in the two level cascade inFIG. 10, the corresponding remote cutter recognition site isincorporated into the previous level cleavage product, but is notrecognized by the remote cutting restriction enzyme at the next leveluntil the remote site on the cleavage product/catalytic primer is madedouble-stranded by polymerase extension opposite the tailing sequence.

The tailing sequence on the catalytic primer can be as little as onenucleotide long, as long as it completes the necessary remoterecognition site. Where a tailing sequence is incorporated into thecatalytic primer, it is not necessary to also include a 3'-end cappingregion on the substrate reagent. Although self-priming and subsequentextension may still occur from folding back of the substrate reagent,this alone cannot create the remote recognition site required forselective cleavage by the cutting agent to generate the undesiredspurious cleavage product. In this event, the only deleterious effect onthe system will be the effective elimination of the self-primedsubstrate reagent from participation in the cascade. Similarly, carrierpriming and subsequent extension fails to create the necessary remoterecognition site for release of cleavage product, leading to the sameresult.

For diagnostic purposes, the final oligonucleotide cleavage product of atarget-initiated amplification cascade is used as a measure of thepresence of target in a test sample. This final "diagnostic"oligonucleotide product may be identical or complementary to a portionof the target sequence, or it may be wholly unrelated in sequence to thetarget. (A single substrate reagent reaction can also be designed togenerate an oligonucleotide product which is different in sequence fromthe target or its complement.) Thus, a diagnostic system according tothe present invention can be designed to be either a targetamplification (where the final oligonucleotide product is identical orcomplementary to target) or a signal amplification (where an unrelatedfinal oligonucleotide product is generated). It will generally bepreferred to employ a signal amplification design, because the finaloligonucleotide product in this type of design cannot participate as acarryover contaminant in future amplification runs. This provides adistinct advantage over target amplification systems such as PCR and LCRwhich are susceptible to these types of contamination problems.

The present invention also provides certain sensitivity and kineticadvantages over traditional diagnostic methods employing signalamplification. These earlier methods typically involve the attachment ofmultiple labels or catalytic moieties to a reporter probe in order toimprove sensitivity. These reporter probes, which bind to the targetnucleic acid sequence, are normally provided in large excess in order toachieve reasonable kinetics. It is, however, necessary to incorporateinto these traditional probing techniques a method for separating excessunhybridized reporter probes from the target-bound probes prior todetecting signal from the target-bound reporter probe. Separation may beachieved through the pre-attachment of target to a solid support, eitherdirectly, or through a previously bound "capture" probe. The reporterprobe becomes attached to the solid support through hybridization withthe pre-attached target, leaving excess unhybridized probe available forremoval through mechanical washing of the solid support. Signal measuredfrom the solid support is indicative of the presence of targetmolecules.

Ideally, reporter probes become attached to the solid support(s) onlythrough hybridization to target molecules. As a practical matter,however, sensitivity limiting background problems occur, becausereporter probes can and do become attached to solid supports throughother means of interaction with the support; i.e., through nonspecificbinding and/or nonspecific hybridization. Signal from nonspecificallybound or nonspecifically hybridized reporter probes limits theachievable sensitivity in a given assay configuration. The sensitivitylimit is reached when the signal from target-hybridized reporter probesapproaches the level of signal produced through the nonspecificallybound or nonspecifically hybridized reporter probes. At lower levels oftarget, where these two types of signals approach the same intensity, itis impossible for an instrument or a human observer to distinguishbetween the two signals, and the presence or absence of target moleculesbecomes meaningless.

The method of the present invention takes advantage of a directcatalytic effect from a substrate reagent that is formed in response tothe presence of target, rather than an indirect effect from catalyticgroups attached to reporter probes. As a result, the present methodavoids the types of background problems which result from nonspecificinteractions caused by the large excess of these reporter probes whichare introduced into the assay. Similarly, the previously mentionedCHA-based diagnostic assay uses target to directly catalyze the cleavageof an excess of labeled detection probe. In one embodiment, for example,the CHA method takes advantage of the catalytic effect of DNA on RNAsubstrates in the presence of RNAse H, previously reported by Berkaweret al, J. Biol. Chem., 248(17), 5914-5921 (1973). In this particular CHAembodiment, the DNA target sequence acts directly as a catalyticco-factor for the RNAse H-induced cleavage of labeled RNA detectionprobes. Once the DNA target hybridizes with the RNA detection probe, theresulting DNA-RNA hybrid becomes a substrate for RNAse H, whichselectively cuts the RNA strand of the duplex. Selective cleavage of theRNA strand lowers the effective T_(m) of the duplex, allowing the RNAfragments melt off of the target sequence. The target may then berecycled to hybridize with another RNA detection probe.

The primary advantage of using target to catalyze signal generation liesin the potential to avoid sensitivity limiting background problemscaused by nonspecific binding and nonspecific hybridization of catalystintroduced in large excess via the reporter probes. The CHA method,however, presents other potential sensitivity limiting features whichare avoided by the method of the present invention. For example, thesignal generated in a CHA reaction is measured by separating a largeexcess of the larger labeled detection probes from the resulting cleavedfragments which also carry the label. The sensitivity of the CHA methodwill therefore be limited to some degree by the efficiency of thisseparation step. Also, spurious, or non-target-catalyzed cleavage of CHAdetection probes by a variety of means will introduce potentialsensitivity limiting background into the assay, even if an efficientseparation step is provided.

Importantly, the cascade method of the present invention relies on insitu synthesis, rather than cleavage of presynthesized probes, toachieve an amplified diagnostic result. The cascade initiation processitself involves the in situ synthesis of a first primed substratereagent in the presence of target sequence using substrate precursors.Although these substrate precursor(s) of the present invention areprovided in large excess with respect to the target, they cannot alone(i.e., without a template) produce a catalytic effect to generate signalor cleavage product. Similarly, because all of the catalytic elements inthe cascade, as well as the final oligonucleotide product responsiblefor generating signal, are generated in situ through assembly fromsmaller elements (rather than through the destruction of larger elementsas in CHA) the method of the present invention is not susceptible to thesensitivity limitations from spurious destruction of substrate moleculesto which the CHA method is prone.

Both the catalytic primer(s) at higher levels in the cascade and thefinal oligonucleotide are indeed, by definition, "cleavage products".However, the extended substrate reagent precursors, required forgeneration of these cleavage products through selective cleavage, do not"pre-exist", but are, as previously noted, created in situ. In contrast,the signal-producing CHA cleavage products are present in large excessin the form of the cleavable detection probe precursors, enablingbackground signal products to be formed through the cleavage of a singlecovalent bond. The final oligonucleotide cleavage product in the methodof the present invention must be assembled from monomericdeoxynucleoside triphosphates. This cannot occur spontaneously, and thusno background signal can result from spurious events.

Further, because the extended substrate reagent cleavage productprecursors in the amplification cascade of the present invention aresynthesized in. situ, it is not necessary to separate the catalyticcleavage product precursors, or extended substrate reagents, byattachment to a solid support, as would be the case for a CHA type ofcascade. Thus, the exponential casqade accumulation of signal-producingoligonucleotide cleavage product in the present invention can proceed ina kinetically unrestrained homogeneous environment Additionally, theunincorporated portion of the large excess of nucleoside triphosphateswhich is used to generate the signal-generating oligonucleotide cleavageproduct of the present invention need not be removed or separated inorder to detect the presence of the resulting target-derivedoligonucleotide product. Sensitivity does not suffer from theunnecessary incorporation of a separation step.

In some instances, it may be desired to use a cascade for purposes ofsynthesizing oligonucleotides. In this case the complementary portion ofthe last substrate reagent will be selected to be complementary to thedesired oligonucleotide product. The remaining substrate reagents in thecascade will typically be designed in reverse order of their appearancein the cascade. In other respects, the cascade will proceedsubstantially as discussed for the amplification cascade used fordiagnostic purposes.

The following examples are provided to aid in the understanding of thepresent invention, the true scope of which is set forth in the appendedclaims. It is understood that modifications can be made in theprocedures set forth, without departing from the spirit of theinvention.

EXAMPLE 1 Synthesis of Oligonucleotide Reagents

The oligonucleotide reagents in the examples were synthesized using anApplied Biosystems model 380B synthesizer (Applied Biosystems, Inc.,Foster City, Calif.) using cyanoethyl phosphoramidites and purified bypolyacrylamide gel electrophoresis as disclosed in International PatentApplication No. 89/02649, which is incorporated herein by reference.

Unmodified oligonucleotide sequences CAT₁ and CAT₁ ' (FIG. 11), CAP₁(FIG. 13), and CAP₂ and CAP₃ (FIG. 15) were synthesized withoutmodification. The preparation of the nucleotide analogs was as follows:

Phosphorothioate-modified oligonucleotide _(S) CAT₁ ' (FIG. 8) wassynthesized as described, with the exception that the phosphate between"C" and "A" as shown in FIG. 8 at position "X" was oxidized for 400seconds with 5% sulfur in pyridine:carbon disulfide (1:2, v/v) insteadof O₂ /iodine. The product was deprotected and purified in the samemanner as the unmodified oligonucleotide reagents.

Phosphorodithioate-modified oligonucleotide _(SS) CAT₁ ' (FIG. 11) wassynthesized in the same manner as the unmodified oligonucleotides, withthe exception that the adenosine 5'- to the modified phosphate shown inFIG. 8 at position "X" was introduced as the5'-dimethoxytrityl-H-benzoyldeoxyadenosine-3'-(N,N-dimethylamino)-(2,4-dichlorobenzylthiyl)-phoshinewhich was prepared according to the method of Caruthers et al, J. Am.Chem. Soc., 111, 2321-2322 (1989). Following condensation with thismodified phosphoramidite, the oligonucleotide was further oxidized withsulfur, as described above. The finished oligonucleotide was deprotectedand removed from the resin using thiophenol followed by ammonia, asdescribed in Caruthers et al, ibid, to produce the desiredphosphorodithioate modified product.

Methylphosphonate-modified oligonucleotides _(Me) CAT₁ ' (FIG. 11) and_(Me) CAT₂ ' (FIG. 15) were synthesized in the same manner as theunmodified oligonucleotides, with the exception that the adenosine 5'-to the modified phosphate was introduced as the methyl phophonamidite,dA-Me Phosphonamidite (Glen research Corporation, Herndon, Va.). Thefinal product was deprotected and removed from silica support bytreating with 30% ammonium hydroxide:pyridine (1:1, v/v) for 4 days at4° C., as described by Noble et al, Nucl. Acids Res., 12(7), 3387-3404(1984).

Methylthiophosphonate oligonucleotide _(SMe) CAT₁ ' (FIG. 11) wasprepared as the methylphosphonate oligonucleotides, with the exceptionthat, after condensation with the modified adenosine, the phosphate wasoxidized with sulfur as described above.

EXAMPLE 2 Screening of Potential Cutting Attenuation Modifications

This example demonstrates the first level of screening for a suitablecutting attenuation modification. The cutting attenuation modificationshould allow for cutting of the unmodified strand of a duplex whileinhibiting cutting of the modified strand of a duplex. In order toscreen for suitable modifications, several DNA duplexes containing a MluI recognition site (ACGCGT) with various phosphate modifications on oneof the strands were prepares A single phosphate modification wasincorporated into the phosphate that participates in cutting by therestriction endonuclease. The sequences used in this evaluation areshown in FIG. 11. The Mlu I recognition site was located asymmetricallywithin the duplex such that resulting different fragment sizes could beused to determine the position of cutting. For example, cutting of theupper strand (CAT₁) yields a 15-mer oligonucleotide fragment and a 9-meroligonucleotide fragment, while cutting of the lower strand (CAT₁ ')yields a 5-mer oligonucleotide fragment and a 19-mer oligonucleotidefragment. Since only the 5'-ends of the duplex were initially labeledwith 32_(P), only the resulting 15-mer oligonucleotide fragment from theupper strand and the 5-mer oligonucleotide fragment from the lowerstrand were expected to appear in autoradiography. Thus, an appropriatecutting attenuation modification located on only the lower strand, CAT₁', was apparent by the appearance of a 15-mer oligonucleotide product,and little or no 5-mer oligonucleotide product upon exposure to the MluI cutting agent.

The following reagents were used:

A 10× reaction buffer was made to contain 100 μg/ml of bovine serumalbumin, 500 mM NaCl, 100 mM Tris.HCl (pH 7.4), 100 mM MgCl₂, and 100 mM2-mercaptoethanol.

Mlu I restriction endonuclease was purchased from New England Biolabs(Beverly, Mass.) at a concentration of 8 units/μl.

Dye reagent was prepared to contain 11 mM EDTA, 83 mM boric acid, 100 mMTris base, 10M urea, 0.02% bromophenol blue, and 0.02% xylene cyanole.

Oligonucleotides CAT₁, CAT₁ ', _(S) CAT₁ ', _(SS) CAT₁ ', _(Me) CAT₁ ',and _(SMe) CAT₁ ' were phosphorylated with γ³² P-ATP(adenosine-5'-triphosphate) obtained from ICN Biomedicals, Inc. (CostaMesa, Calif.), and T4 polynucleotide kinase (New England Biolabs) to aspecific activity of approximately 7,000 Ci/mmole. Theseoligonucleotides were then used to adjust the specific activity of therespective unlabeled oligonucleotides to approximately 1,250 cpm/pmole.

Two independent sets of reactions were established to evaluatemodifications of the phosphate as potential cutting attenuationmodifications. Reaction Set A compared a phosphorothioate (Reaction 3)and a phosphorodithioate (Reaction 2) modification to the naturalphosphate (Reaction 1). Reaction Set B compared a methylthiophosphonate(Reaction 6) and a methylphosphonate (Reaction 5) to the naturalphosphate (Reaction 4). Each reaction was set up to contain a finalvolume of 25 μl of 1× reaction buffer and contained 20 pmole of CAT₁,and 16 units of Mlu I restriction enzyme. In addition to the above, thereactions also contained the following:

    ______________________________________    Reaction Set A    Reaction 1:      20 picomoles of CAT.sub.1 '    Reaction 2:      20 picomoles of .sub.SS CAT.sub.1 '    Reaction 3:      20 picomoles of .sub.S CAT.sub.1 '    Reaction Set B    Reaction 4:      20 picomoles of CAT.sub.1 '    Reaction 5:      20 picomoles of .sub.Me CAT.sub.1 '    Reaction 6:      20 picomoles of .sub.SMe CAT.sub.1 '    ______________________________________

The Mlu I restriction enzyme was added to the reactions after annealingof the oligonucleotides by heating to 90° C. for 2 minutes, followed bycooling at room temperature for 5 minutes. The cutting reactions wereallowed to incubate for 1 hour at 37° C. The reactions were thenquenched by adding 25 μl of dye reagent and heating to 90° C. for 2minutes, followed by cooling to room temperature. The reaction productsin the samples were analyzed by separation using 15% denaturingpolyacrylamide gel electrophoresis (PAGE), followed by autoradiography.As shown in FIG. 12A, the natural phosphate duplex (Reaction 1, Lane 1)shows the expected cutting products, namely a 15-mer and a 5-meroligonucleotide product, in roughly the same ratio.

It should be noted that in addition to these products, there is alsosome remaining 24-mer starting material that does not cut. This is acommon occurrence, and is attributable to the fact that a certainpopulation of synthetic oligonucleotides are not biologically active dueto subtle modifications or incomplete deprotection, as earlierdiscussed. The phosphorodithioate modification (Reaction 2, Lane 2)shows both (15-mer and 5-mer) cleavage products, indicating that it isnot an appropriate modification for this restriction site. It isinteresting to note that this modification inhibits the overall cuttingof both strands, probably by disrupting the substrate affinity for thisenzyme. Similarly, the phosphorothioate modification (Reaction 3, Lane3) was also ruled out as a cutting attenuation modification for use incombination with the Mlu I cutting agent, due to its apparent inabilityto impart cutting resistance.

In contrast, as shown in FIG. 12B, both the methylphosphonate (Reaction5, Lane 5) and the methylthiophosphonate (Reaction 6, Lane 6) providevery efficient cutting attenuation, as demonstrated by the presence ofonly a trace of the 5-mer cleavage product. Additionally, the duplexesstill have good affinity for the enzyme as indicated by the formation ofa comparable degree of 15-mer product, as compared to the unmodifiedduplex (Reaction 4, Lane 4). Thus, either of these cutting attenuationmodifications meet the first requirement for cutting resistance, bytheir ability to attenuate cutting without interfering with substrateaffinity and specificity.

EXAMPLE 3 Polymerase Extension Through Potential Cutting AttenuationModifications

This example demonstrates a screening technique for a second requirementin selecting a cutting attenuation modification, namely the ability of apolymerase to extend through the modification. In order to address thisrequirement for the methylphosphonate and the methylthiophosphonatemodifications, a 9-mer oligonucleotide sequence (CAP₁), complementary tothe 3'-end of the CAT₁ ' control sequence was synthesized for use as acatalytic primer in this extension example. As shown in FIG. 13, thiscatalytic primer is complementary to CAT₁ ', _(Me) CAT₁ ', and _(SMe)CAT₁ ', and, in the presence of polymerase, and dNTP's, should be ableto extend to form a 24-mer product, provided the cutting attenuationmodification does not interfere with the polymerase activity. Theproducts were labeled, for subsequent visualization, by incorporatingα³² P-dATP into the polymerase extension reactions.

The following reagents were used:

10× Extension Buffer was 100 mM Tris.HCl (pH 7.4, 100 mM MgCl₂, 100 mM2-mercaptoethanol, and contained 100 μg/ml bovine serum albumin.

DNA Polymerase I Large Fragment (Klenow Fragment) was purchased at aconcentration of 8 units/μl from New England Biolabs.

Deoxyadenosine-5'-triphosphate alpha-³² P! (α³² P-dATP) was purchasedfrom ICN Biomedicals, Inc. at a specific activity of 3,000 Ci/mmole.

Deoxynucleoside-5'-triphosphates (dATP, dCTP, dGTP, and dTTP) wereobtained as part of the Perkin Elmer Cetus (Norwalk, Conn.) GeneAmp™ DNAAmplification Reagent Kit.

Dye reagent was the same as used in Example 2.

The deoxyadenosine-5'-triphosphate used in the examples was adjusted toa specific activity of approximately 2,000 CPM/pmole by mixing theappropriate amount of α³² P-dATP and dATP. In addition, the ³² P-labeledCAP₁ from Example 2 was used as a 24-base marker in gel electrophoresis.

All reactions were run in a final volume of 10 μl of 1× ExtensionBuffer, and were 100 mM in each dNTP and contained 4 pmoles of CAP₁ and1.0 units of Klenow Fragment Polymerase. In addition to the above, thereactions also contained the following:

Reaction 1: 2.0 picomoles of CAT₁ '

Reaction 2: 2.0 picomoles of _(Me) CAT₁ '

Reaction 3: 2.0 picomoles of _(SMe) CAT₁ '

The Klenow Fragment polymerase was added to the reactions last, afterannealing by heating to 90° C. for 2 minutes, followed by cooling toroom temperature for 5 minutes. The extension reactions were allowed torun for 15 minutes at room temperature, and then quenched by theaddition of 10 μl of dye reagent and heating to 90° C. for 2 minutes,followed by cooling again to room temperature. The products, along witha ³² P-labeled 24-mer oligonucleotide which was used as a marker, wereseparated by running on 15% denaturing PAGE and visualized byautoradiography.

As shown in FIG. 14, all three CAT sequences serve as templates to formthe 24-mer extended substrate reagent. Reaction 1 (Lane 1) formed aclean 24-mer fragment, while the modified template sequences in Reaction2 (Lane 2) and Reaction 3 (Lane 3) formed the 24-mer extended substratereagent and some less than fully extended products. Thus, both of thesephosphate modifications meet the second requirement for the cuttingattenuation modification with the exception that, in addition to thefully extended product, they also formed some less than fully extendedproduct. The methylphosphonate modification was selected for the cuttingattenuation modification in subsequent examples, because it is somewhatmore convenient to prepare the methylphosphonate than themethylthiophosphonate modified sequence.

EXAMPLE 4 Synthesis of an Oligonucleotide Product Using aMethylphosphonate-modified Substrate Reagent

The final requirement for an acceptable cutting attenuation modificationis that it operate satisfactorily with the cutting agent and thepolymerizing agent simultaneously in the same reaction environment. Inorder to test whether the methylphosphonate cutting attenuationmodification would perform satisfactorily under these conditions,substrate reagent (_(Me) CAT₂ ') and catalytic primer (CAP₂), shown inFIG. 15, were both treated with Klenow polymerase and Mlu Isimultaneously. If both the cutting process and the extension processwork in the same reaction, one should observe an accumulation of a10-mer oligonucleotide cleavage product CAP₃. Additionally, thecatalytic primer CAT₂ was titrated down in concentration in this exampleto confirm that production of CAP₃ was indeed due to the presence ofcatalytic primer. Label was incorporated into the products forsubsequent visualization by using the radioactive α³² P-dATP asdescribed in Example 3.

In order to quantitate the accumulation of CAP₃ cleavage product, thesame reactions were run using polymerase alone, without the cuttingagent. In this case, all primed substrate reagent will extend to form asingle copy of extended substrate reagent. The number of cycles toproduce the observed amount of cleavage product (CAP₃) where bothreagents are present could then be estimated by comparison to thissignal, representing a single copy.

The following reagents were used:

Reaction Buffer, Dye Reagent, and Mlu I restriction endonuclease werethe same as in Example 2.

Oligonucleotides CAP₃ and _(Me) CAT₂ ' were labeled with γ³² P-dATP andpolynucleotide kinase to a specific activity of approximately 7,000Ci/mmole for used as markers in identifying reaction products in gelelectrophoresis.

Deoxynucleoside triphosphates and Klenow Fragment polymerase were thesame as in Example 3.

All reactions were run in a final volume of 15 μl of 1× Reaction Buffer,and were 67 μM in each dNTP and contained 4 pmoles of _(Me) CAT₂ ' and1.0 unit of Klenow polymerase. In addition, the reactions alsocontained:

Reaction 1: 2.0 pmoles CAP₂

Reaction 2: 2.0 pmoles CAP₂ +8.0 units Mlu I

Reaction 3: 0.2 pmoles CAP₂

Reaction 4: 0.2 pmoles CAP₂ +8.0 units Mlu I

Reaction 5: 0.02 pmoles CAP₂

Reaction 6: 0.02 pmoles CAP₂ +8.0 units Mlu I

Reaction 7: 0.0 pmoles CAP₂

Reaction 8: 0.0 pmoles CAP₂ +8.0 units Mlu I

The Klenow polymerase and/or Mlu I restriction enzyme were added last tothe reaction mixtures after annealing by heating to 90° C. for 2 minutesfollowed by cooling to room temperature for 5 minutes. The enzyme(s)were added to the reaction mixtures in a volume of 5 μl, bringing thetotal volume to 15 μl. The reactions were allowed to incubate at 45° C.for 3.5 hours and quenched by adding 15 μl of dye reagent and heating to90° C. for 2 minutes followed by cooling at room temperature. Thereactions, along with a ³² P-labeled 10-mer and a ³² P-labeled 25-meroligonucleotide marker, were separated by 15% denaturing PAGE and theproducts were visualized by autoradiography.

The results shown in FIG. 16 suggest that multiple copies of thecleavage product (CAP₃) are indeed being formed. Reaction 1 (Lane 1),where no cutting agent was present, shows a 25-mer oligonucleotideproduct that represents a single copy of extended substrate reagent. Incontrast, Reaction 2 (Lane 2) shows (in addition to the extendedsubstrate reagent) an accumulation of the 10 base cleavage product CAP₃.It is interesting to note that, in addition to the expected 10-mercleavage product, there is also an accumulation of shorter cleavageproducts, namely a 9-mer, 8-mer, and a 7-mer. These incomplete cleavageproducts most likely result from cleavage of the less than fullyextended substrate reagents as observed in Example 3. Scanning laserdensitometry using an UltroScan™ XL laser densitometer (Pharmacia LKBBiotech, Inc., Piscataway, N.J.) indicates that there is approximately25 times more cleavage product than extended substrate reagent. Thistakes into account that the extended substrate reagent contains tworadioactive adenosine moieties, and the cleavage products contain onlyone radioactive adenosine moiety. It is also apparent that the amount ofcleavage product, as shown in Lanes 2, 4, 6, and 8 (Reactions 2, 4, 6,and 8, respectively) is formed in proportion to the amount of CAP₂catalytic primer. As predicted, Reaction 8 shown that no 10-mer cleavageproduct CAP₃ is formed by substrate reagent in the absence of CAP₂catalytic primer.

EXAMPLE 5 Cascade Production of Oligonucleotides

The following example demonstrates the use of a two level cascade toproduce an oligonucleotide cleavage product according to the schemeshown in FIG. 17. In this example, the first level of the two levelcascade was allowed to cycle either by itself or in the presence of thesecond level substrate reagent. In addition, the first level substratereagent was titrated down in concentration to confirm that any cleavageproducts were produced in response to its presence. As shown in FIG. 17,the two level cascade scheme used in this example was designed toproduce a 9-mer first level cleavage product (CAP₂) and a 10-mer secondlevel cleavage product (CAP₃). The size difference between the twocleavage products enabled the consequent differences in mobility of theproducts to be used as a means for differentiation using denaturing PAGEseparation. All products were labeled in situ through incorporation of³² P-labeled dATP, as in Example 3 and Example 4.

The following reagents were used:

Oligonucleotides CAP₂, CAP₃, _(Me) CAP₂, and _(Me) CAP₁ ' werephosphorylated on their 5'-ends using γ³² P-dATP (ICN Biomedicals, CostaMesa, Calif.) and T4 polynucleotide kinase (New England Biolabs, Inc.)at a specific activity of approximately 7,000 Ci/mmole. Theseoligonucleotides were used as markers to characterize the resultingproducts which were analyzed by PAGE and autoradiography.

All other reagents were as described in Example 4.

All reactions were run in a final volume of 15 μl of 1× reaction buffercontaining 4 pmoles of catalytic primer CAP₁, 1.0 unit of Klenowpolymerase, and 8.0 units of Mlu I and were 66.7 μM in each dNTP (thedATP was adjusted to a specific activity of approximately 2,000CPM/pmole). In addition to the above, the reactions also contained thefollowing:

    ______________________________________                .sub.Me CAT.sub.1 '                        .sub.Me CAT.sub.2 '    ______________________________________    Reaction 1:   250 fmoles                            4.0 pmoles    Reaction 2:    25 fmoles                            4.0 pmoles    Reaction 3:   2.5 fmoles                            4.0 pmoles    Reaction 4:   0.0 fmoles                            4.0 pmoles    Reaction 5:   250 fmoles                            0.0 pmoles    Reaction 6:    25 fmoles                            0.0 pmoles    Reaction 7:   2.5 fmoles                            0.0 pmoles    Reaction 8:   0.0 fmoles                            0.0 pmoles    ______________________________________

The enzymes were added last to the reactions after annealing by heatingto 90° C. for 2 minutes followed by cooling to room temperature for 5minutes. Following addition of the enzyme, the reactions mixtures wereincubated at 45° C. for 2 hours and 40 minutes. After incubation withthe enzyme, the reactions were quenched by the addition of 15 μl of dyereagent and heated to 90° C. for 2 minutes, and then allowed to cool toroom temperature. The resulting oligonucleotide products were analyzed,using standard techniques, by running the reaction mixtures and markerson 15% denaturing PAGE, followed by visualization on autoradiography.

As shown in FIG. 18, the desired 10-mer oligonucleotide cleavage productCAP₃ from the cascade was formed in Reactions 1, 2, and 3 (Lanes 1, 2,and 3, respectively), as determined by its similar mobility with respectto the 10-mer oligonucleotide marker (Lane M). Reaction 4 (Lane 4),which contains no first substrate reagent, showed no signs of cleavageproduct CAP₃, even upon prolonged radiography. This confirms that thedesired cascade cleavage product CAP₃ in Reactions 1, 2, and 3 wasproduced specifically in response to the cascade. The cleavage productCAP₂ was also produced in a quantity proportional to the amount ofstarting substrate reagent _(Me) CAT₁ '.

Further confirmation that the desired 10-mer cleavage product was indeedan end product of the cascade was obtained by analyzing the data for theReactions run in the absence of the second level substrate reagent _(Me)CAT₂ ' (Reactions 5-8). Reaction 5 (Lane 5) shows that no 10-meroligonucleotide product was formed in the absence of the secondsubstrate reagent. The expected first level cleavage product was formedat a low level with the primary cleavage products from the first levelof the cascade being the 8-, 7-, and 6-mer oligonucleotide products,which were apparently generated as the product of incomplete extensionof the first level primed substrate reagent.

These "incomplete" first level cleavage products also appear in thecorresponding two level cascade (Reaction 1, Lane 1) to approximatelythe same degree that they appear in the one substrate reagent reaction.This could be due to the fact that these "incomplete" cleavage productsare not long enough to initiate priming of polymerase extension of thesecond level substrate reagent. It is also possible that if one were todesign the system to generate longer cleavage products (e.g., a 12-meroligonucleotide product), the "incomplete" cleavage products from thisreaction (e.g., 11-, 10-, and 9-mer oligonucleotide products) would becapable of priming the next level of the cascade, thereby improvingefficiency and, consequently, the yield of the desired end product.

In the present example, if one were to assume that only the full length9-mer cleavage product from the first level of the cascade is capable ofpriming the second level substrate reagent, there is an approximate100-fold benefit in sensitivity achievable by using the second level ofthe cascade. This calculation is based on the fact that the 9-meroligonucleotide cleavage product formed in response to 250 femtomoles of_(Me) CAT₁ ' in the one substrate reagent reaction (Reaction 5, Lane 5)is being formed at approximately the same level as the 10-meroligonucleotide cleavage product formed in response to only 2.5femtomoles of _(Me) CAT₁ ' in the two level cascade (Reaction 3, Lane3).

EXAMPLE 6 Target-Dependent Synthesis of Substrate Reagent UsingSubstrate Precursors and Ligase

This example demonstrates the synthesis of a completed substrate reagentfrom two substrate precursors in the presence of a target sequence, asillustrated in FIG. 4. The target sequence in this example serves as atemplate to bring the two precursor sequences (one of which contains aCAM) into contiguous proximity so that they can be covalently joined bythe enzyme E. coli ligase. The oligonucleotide sequences used in thisexample are shown in FIG. 19.

The presence of the CAM near the ligation junction of oligonucleotide_(Me) SP₁ ' was seen as a potential source of interference with theaction of ligase in covalently joining _(Me) SP₁ ' to SP₂ ' in this typeof target-initiated synthesis of substrate reagent. It was thereforedecided to simultaneously evaluate a control system (employing wild typeoligonucleotide SP₁ '; i.e., not containing the CAM) in order toevaluate the influence of this particular CAM modification on therequired ligation event. However, it should be noted that, while theligation of SP₁ ' to SP₂ ' serves as an effective gauge of ligationefficiency, the resulting product is not an effective substrate reagentbecause it does not contain the necessary CAM.

Oligonucleotides TIC₁ (Target Sequence), SP₁ ' (Control SubstratePrecursor 1'), and SP₂ ' (Substrate Precursor 2') were synthesized andpurified as unmodified oligonucleotides, as described in Example 1.

Methylphosphonate-modified oligonucleotide _(Me) SP₁ ' (SubstratePrecursor 1') was synthesized and purified, as described in Example 1.The single methylphosphonate linkage, 3' to the adenosine, as shown inFIG. 19, was introduced to serve as a cutting attenuation modification(CAM) for Mlu I restriction endonuclease.

Adenosine 5'-triphosphate (ATP), used to phosphorylate oligonucleotideSP₂ ', was purchased from Sigma Chemical Company (St. Louis, Mo.).

Radioactive γ³² P-ATP, at a specific activity of approximately 7,000Ci/mmole, was purchased from ICN Biomedicals, Inc.

T₄ polynucleotide kinase, at a concentration of 10 units/μl, waspurchased from New England Biolabs, Inc.

The enzyme E. coli ligase, at a concentration of 7 units/μl, waspurchased from Boehringer Mannheim Corporation (Indianapolis, Ind.).

Nicotinamide adenine dinucleotide (NAD) was purchased from SigmaChemical Company.

Oligonucleotides TIC₁, SP₁ ', and _(Me) SP₁ ' were phosphorylated ontheir 5'-ends to a specific activity of approximately 7,000 Ci/mmoleusing γ³² P-ATP and T₄ polynucleotide kinase. Excess ATP was separatedfrom the labeled oligonucleotides by passing each reaction mixture overa Sephadex® G 50/50 column. Labeled TIC₁ ' was used as a gel marker,while labeled oligonucleotides SP₁ ' and _(Me) SP₁ ' were used toincorporate labels into reaction products for visualization followinggel electrophoresis.

Oligonucleotide SP₂ ' was phosphorylated using T₄ polynucleotide kinaseand γ³² P-ATP (diluted with ATP to a specific activity of approximately1.0 Ci/mmole). The radioactive label was incorporated at a low specificactivity as a means of following the phosphorylation efficiency. ExcessATP was separated from the labeled oligonucleotide by passing thereaction mixture over a Sephadex® G 50/50 column using triethylammoniumbicarbonate as an eluant. Fractions containing the oligonucleotide werecombined and then evaporated and resuspended in TE (Tris.HCl/EDTA).Scintillation counting showed that this oligonucleotide wasphosphorylated quantitatively.

All ligation reactions (20 μl final volume) were run in 50 mM Tris (pH7.6), 6.6 mM MgCl₂, 6.6 mM DTT, and 66 μM NAD, and additionallycontained 0.5 mg/ml of BSA and 0.46 units of E. coli ligase. Thereactions also contained the following:

    ______________________________________              SP.sub.1 '*                       .sub.Me SP.sub.1 '*                                 TIC.sub.1                                        SP.sub.2 '    Reaction  (pmoles) (pmoles)  (pmoles)                                        (pmoles)    ______________________________________    1         0.0      4.8       0.0    5.0    2         0.0      4.8       0.0    5.0    3         0.0      4.8       2.0    5.0    4         0.0      4.8       2.0    5.0    5         0.0      4.8       0.0    0.0    6         0.0      4.8       0.0    0.0    7         4.8      0.0       0.0    5.0    8         4.8      0.0       0.0    5.0    9         4.8      0.0       2.0    5.0    10        4.8      0.0       2.0    5.0    11        4.8      0.0       0.0    0.0    12        4.8      0.0       0.0    0.0    ______________________________________     *Specific activity of SP.sub.1 ' and .sub.Me SP.sub.1 ' = 7,000 Ci/mmole

The reaction mixtures were heated to 90° C. for 2 minutes, and thencooled to room temperature to effect annealing of the substrateprecursors to the target sequence. (The ligase was added to eachreaction mixture after the annealing step, resulting in a final volumeof 20 μl.) After 1 hour at room temperature, 20 μl of dye reagent wasadded to each reaction, and the reaction tubes were then heated to 90°C. for 2 minutes to inactivate the enzyme. The reaction products werethen analyzed by running the samples on denaturing 15% PAGE, followed byvisualization using a PhosphorImager™ data collection device programmedwith ImageQuant™ software (instrument and software from MolecularDynamics, Sunnyvale, Calif.). The PhosphorImager™ device replaces thetraditional use of photographic film to create a record of theseparation data on the gel (i.e., autoradiography). The ImageQuant™software, in combination with the PhosphorImager™ device, provides both:(1) qunatitative data, otherwise achieved through the use ofdensitometry; and, (2) a graphic "printout" representative of the imageachieved in a traditional autoradiogram.

A printout of the data from the PhosphorImager™ is shown in FIG. 20.Lanes 1-12 correspond to Reactions 1-12, respectively. Radioactive TIC₁(30-mer) was loaded on either side of the gel for use as a marker. Asshown in Lanes 3 and 4, the expected ligation product (30-mer) wasformed from substrate precursors SP₂ ' and _(Me) SP₁ ' in the presenceof target sequence TIC₁. In the case where both substrate precursorswere wild type oligonucleotides (SP₂ ' and SP₁ ', Lanes 9 and 10,respectively) the 30-mer product formed in approximately the same yield.This suggests that the CAM-containing oligonucleotide _(Me) SP₁ ' servesas an effective substrate for E. coli ligase despite the presence of themethylphosphonate internucleotide linkage in close proximity to theligating junction. No 30-mer ligation product was formed in controlreactions where either: (1) the target was absent (Lanes 1, 2, 7, and8); or, (2) the target and substrate precursor SP₂ ' was absent (Lanes5, 6, 11, and 12). This confirms that the product is formed as a directresult of the presence of target in the reaction mixtures.

EXAMPLE 7 Target Dependent Synthesis of Substrate Reagent Using aSubstrate Precursor and Polymerase

This example demonstrates yet another type of target-initiated synthesisof a completed substrate reagent, wherein the substrate reagent isformed from a single substrate precursor, containing a CAM, in thepresence of an excess of dNTP's, polymerase, and a target sequence, asillustrated in FIG. 5. The target sequence in this example serves astemplate for hybridization and extension of the modified substrateprecursor _(Me) SP₃ ' in the presence of the dNTP's and a polymerase(Klenow), as shown in FIG. 21. Once synthesized, this substratereagent-target duplex can be cut in the presence of Mlu I restrictionendonuclease to produce an activated substrate reagent and anaccumulation of oligonucleotide cleavage product, as demonstrated inExamples 8 and 9. The oligonucleotide sequences used in this example areshown in FIG. 21.

Similarly to the previous Example 6, the location of a methylphosphonateinternucleotide linkage near the point of polymerase extension from theCAM-modified substrate precursor was seen as potentially interferingwith the required action of the enzyme, in this case a polymerase. Inorder to evaluate the influence of the methylphosphonate CAMmodification on polymerase extension, the corresponding wild typeoligonucleotide SP₃ ' was synthesized for use as a control in thecomparison of extension efficiency with the modified substrateprecursor.

Oligonucleotides TIC₁ and ³² P-TIC₁ (7,000 Ci/mmole, used as a gelmarker) were the same as in Example 6.

DNA polymerase I Large Fragment (Klenow), at a concentration of 5units/μl, was purchased from New England Biolabs, Inc.

Deoxyadenosine 5'-triphosphate alpha-³² P! (α³² P-dATP) anddeoxynucleoside-5'-triphosphates (dATP, dCTP, dGTP, and dTTP) were thesame as used in Example 3. The dATP used in this example was adjusted toa specific activity of approximately 4,000 dpm/pmole by mixing theappropriate amount of α³² P-dATP and dATP.

Oligonucleotide SP₃ ' (Control Substrate Precursor 3') andmethylphosphonate-containing oligonucleotide _(Me) SP₃ ' (SubstratePrecursor 3') were synthesized and purified, as described in Example 1.

A 10× reaction buffer was prepared as 500 mM Tris.HCl (pH 7.9), 100 mMMgCl₂, 1M NaCl, and 10 mM DTT.

All polymerase extension reactions were run in a final volume of 15 μlof 1× reaction buffer and contained each of the dNTP's at aconcentration of 66.7 μM, as well as 1 unit of Klenow, and 2 pmoles ofTIC₁ (unlabeled). The products resulting from polymerase extension weredesigned to be radioactive (from incorporation of radioactive dATP(4,000 dpm/pmole) during polymerase extension) to provide a means forsubsequent visualization. The reactions also contained the following:

    ______________________________________                   SP.sub.3 '                            .sub.Me SP.sub.3 '    Reaction       (pmoles) (pmoles)    ______________________________________    1              0.0      0.0    2              0.0      0.0    3              0.0      0.4    4              0.0      0.4    5              4.0      0.0    6              4.0      0.0    ______________________________________

The reaction mixtures were heated to 90° C. for 2 minutes, and thencooled to room temperature to effect annealing of the primers to thetarget sequences. (Klenow polymerase, in a volume of 5 μl, was added toeach reaction after this step, resulting in a final reaction volume of15 μl.) The extension reactions were allowed to proceed for 1 hour at37° C. The reactions were stopped by adding 15 μl of dye reagent to eachreaction, followed by heating to 90° C. for 2 minutes and then coolingto room temperature. The reaction products were analyzed by running thereactions on denaturing 15% PAGE, followed by visualization of theradioactive products using a PhosphorImager™ with ImageQuant™ software.

A printout of the data from the PhosphorImager™ is shown in FIG. 22.Lanes 1-6 correspond to reactions 1-6, respectively. Radioactive TIC₁(30-mer) was loaded on either side of the gel as a marker. As seen inLanes 3, 4, 5, and 6, the expected 30-mer extension product was formedequally well with either the wild type control substrate precursor (SP₃', Lanes 5 and 6) or the methylphosphonate-containing modified primersubstrate precursor (_(Me) SP₃ ', Lanes 3 and 4). Reactions in theabsence of primer (Lanes 1 and 2) show no sign of 30-mer extensionproduct, confirming that the products observed in Lanes 3-6 are indeedextension products from the respective substrate precursors.

EXAMPLE 8 Target-Dependent Synthesis of Oligonucleotides Using KlenowPolymerase

This example demonstrates the preparation of a completed substratereagent (using a single substrate precursor), followed byoligonucleotide synthesis. The production of oligonucleotide productsfrom the completed substrate reagent occurs as a result of theadditional presence of a cutting agent (Mlu I) to simultaneouslygenerate both the activated substrate reagent and cleavage product, asillustrated in FIG. 23. The target sequence TIC₁ serves as a templatefor substrate precursor (_(Me) SP₃ ') which extends in the presence ofKlenow polymerase and dNTP's to form the completed substrate reagent(_(Me) CAT₃ '). Radioactive dATP was employed in this reaction as ameans to visualize the completed substrate reagent and cleavage productsafter PAGE.

The resulting substrate reagent duplex is designed to be cleaved only onthe upper strand, by cutting agent Mlu I, thus releasing oligonucleotideproduct (CAP₄, a 10-mer) and a target-activated substrate reagent, whichcan then be cycled to generate additional CAP₄ oligonucleotide product.The yield of product in this example can be estimated by comparison ofthe radioactive signal of the desired 10-mer CAP₄ oligonucleotideproduct (three radioactive phosphates) to that of the 30-mer substratereagent (five radioactive phosphates).

It should be noted that, where the target sequence is flanked byadditional nucleotides on its 5'- and 3'-ends (as is expected with atarget obtained from a clinical sample), the resulting extension productwill also contain complementary flanking sequence. Consequently, boththe activated substrate reagent and the first oligonucleotide productreleased following the initial cutting step in this type of targetinitiation will be of an undefined length, as illustrated in FIG. 5. Theundefined length of the activated substrate reagent will not affect itsfunction in the synthesis of subsequent oligonucleotide cleavageproducts, which will then be of the desired defined length.

Oligonucleotide TIC₁ (target sequence), ³² P-TIC₁ (7,000 Ci/mmole, usedas a gel marker), _(Me) SP₃ ', Klenow polymerase, dNTP's, α³² P-dATP,and 10× reaction buffer were the same as in Example 7.

Radioactive γ³² P-ATP and T₄ polynucleotide kinase were the same as inExample 6.

Oligonucleotide CAP₄ (10-mer), used as a gel marker was synthesized andpurified, as described in Example 1.

The marker oligonucleotide CAP₄ was phosphorylated on its 5'-end to aspecific activity of approximately 7,000 Ci/mmole using γ³² P-ATP and T₄polynucleotide kinase. Excess ATP was separated from the labeledoligonucleotide by passing the reaction over a Sephadex® G 50/50 column.

Mlu I restriction endonuclease, at a concentration of 10 units/μl, waspurchased from New England Biolabs, Inc.

The dATP used in this example was adjusted to a specific activity ofapproximately 400 dpm/pmole by mixing the appropriate amount of the α³²P-dATP and dATP.

All reactions were run in a final volume of 20 μl of 1× reaction buffer,and contained each dNTP at a concentration of 50 μM, 1 unit of Klenow,10 units of Mlu I, and 10 pmoles of _(Me) SP₃ '. The reaction mixturesalso contained the following:

    ______________________________________                  Target                  TIC.sub.1           Reaction                  (pmoles)    ______________________________________           1      10.0           2      10.0           3      3.3           4      3.3           5      1.1           6      1.1           7      0.0           8      0.0    ______________________________________

The reaction mixtures were heated to 90° C. for 2 minutes, and thencooled to room temperature to effect annealing of the primers to thetarget sequences. (Klenow polymerase and Mlu I restriction enzyme wereadded as a mixture to the reaction mixtures in a volume of 5 μl afterthis step, resulting in a final reaction volume of 20 μl.) The reactionswere set up in 1.5 ml screw top tubes containing an "o-ring" in the cap(available from Starstedt, Inc., Newton, N.C.) such that the reactionscould be submerged in a constant temperature water bath to preventevaporation. The synthesis reactions were allowed to run overnight (16hours) at 40° C., and then stopped by adding 20 μl of dye reagent toeach reaction mixture. This was followed by heating to 90° C. for 2minutes, and then cooling to room temperature. The resulting reactionproducts were analyzed by running the reactions on denaturing 20% PAGE,followed by visualization of the radioactive products using aPhosphorImager™ with ImageQuant™ software.

A printout of the data from the PhosphorImager™ is shown in FIG. 24.Lanes 1-8 correspond to reactions 1-8, respectively. A mixture of ³²P-labeled TIC₁ and ³² P-labeled CAP₄ was loaded on either side of thegel to serve as markers (30-mer and 10-mer, respectively). The expected30-mer substrate reagent and the corresponding 10-mer oligonucleotideproduct (CAP₄) were formed in all reactions containing target (TIC₁ ;e.g., Lanes 1-6). In contrast, neither product appeared in the "notarget" control reactions (Lanes 7 and 8). The 30-mer substrate reagentgenerated in this reaction runs slightly slower than the 30-mer TIC₁marker. This is to be expected, because the substrate reagent containsone fewer negative charge than the TIC₁ target, or marker, due to thepresence of the single methylphosphonate modification initially presentin the substrate precursor _(Me) SP₃ '.) Additionally, the amount ofproduct (CAP₄) produced in the reactions corresponds to the amount ofstarting sequence.

The number of oligonucleotides synthesized by each activated substratereagent can be estimated by comparison of the intensity of the productsignal (CAP₄, containing 3 radioactive phosphates) to that of thesubstrate reagent (_(Me) CAT₃ ', containing 5 radioactive phosphates)and normalizing the signals, with respect to the number of radioactivephosphates. Based on quantitation using the ImageQuant™ software, 20 to50 oligonucleotides were synthesized for each target molecule in thisexample.

EXAMPLE 9 Target-Dependent Synthesis of Oligonucleotides Using TaqPolymerase

This example demonstrates the target-initiated synthesis of anoligonucleotide (as described in Example 8) using Taq polymerase insteadof Klenow polymerase.

All of the reagents used in this example were the same as used inExample 8, with the exception of the following.

Taq polymerase was used instead of Klenow polymerase. AmpliTaq™ DNApolymerase was obtained from Perkin-Elmer/Cetus at a concentration of 5units/μl.

The dATP in this example was adjusted to a specific activity ofapproximately 4,000 dpm/pmole by mixing the appropriate amounts of α³²P-dATP and dATP.

All reactions were run in a final volume of 20 μl of 1× reaction bufferand contained each dNTP in a concentration of 50 μM, 2.5 units ofAmpliTaq™ DNA polymerase, 10 units of Mlu I, and 2.0 pmoles of substrateprecursor, _(Me) SP₃ '. In addition, the reactions mixtures alsocontained the following:

    ______________________________________                  Target                  TIC.sub.1           Reaction                  (pmoles)    ______________________________________           1      1.0           2      1.0           3      0.33           4      0.33           5      0.11           6      0.11           7      0.0           8      0.0    ______________________________________

The synthesis reactions were set up and allowed to run overnight (16hours), as described in Example 8.

A printout of the data from the PhosphorImager™ is shown in FIG. 25.Lanes 1-8 correspond to reactions 1-8, respectively. A mixture of ³²P-labeled TIC₁ and CAP₄ was loaded on the right side of the gel to serveas markers (30-mer and 10-mer, respectively). As seen in Lanes 1-6, theexpected 30-mer substrate reagent (slightly slower moving than the30-mer marker, as previously explained in Example 8) is formed in allreactions containing target sequence (TIC₁). Additionally, a very strongsignal can be seen in the area of the 10-mer CAP₄ marker. In contrast,no signals were produced from the "no target" control reactions, Lanes 7and 8. The visible amount of 11-mer product, also produced in theoligonucleotide synthesis reactions (e.g., Lanes 1 and 2), is probablydue to the fact that Taq polymerase is known to add one or morenucleotides onto the 3'-ends of extension products beyond the end of thetemplate. Based on quantitation derived from ImageQuant™ software, 160to 240 oligonucleotide products were synthesized for every targetmolecule present in these reactions.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 10    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 bases    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    CCGAGAACGAGATTACGCGTATAA24    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    CCGAGAACGAGATTA15    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 bases    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    CGCGTAATCTCGTTCTCGG19    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 bases    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    CGCGTATAACTCTGACGCGTTATTT25    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 bases    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    CGCGTATAACTCTGA15    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 bases    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    GACGTATGCTACCTGATAGACGCGTAATAT3    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 bases    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    GTCTATCAGGTAGCATACGTC21    (2) INFORMATION FOR SEQ ID NO: 8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 bases    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    ATATTACGCGTC12    (2) INFORMATION FOR SEQ ID NO: 9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 bases    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    CGCGTAATAT10    (2) INFORMATION FOR SEQ ID NO: 10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 bases    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    GACGTATGCTACCTGATAGA20    __________________________________________________________________________

What is claimed is:
 1. A diagnostic kit comprising a nucleic acidtemplate sequence precursor, an excess of deoxynucleoside triphosphates,an agent for polymerization, and a nuclease, wherein said templatesequence precursor has a cutting attenuation modification that preventscleavage of the template precursor during selective nuclease cleavage ofan extension of the template sequence and wherein said template sequenceprecursor is complementary to a portion of a target sequence.
 2. Adiagnostic kit comprising a first nucleic acid template sequenceprecursor, a second nucleic acid template sequence precursor, a ligaseand a nuclease, wherein one of said first or second template sequenceprecursors has a cutting attenuation modification that prevents cleavageof the template precursor during selective nuclease cleavage of anextension of the template sequence and wherein both of said templatesequence precursors are complementary to contiguous portions of a targetsequence.